MAT). The human fungal pathogenic and basidiomycetous yeast Cryptococcus neoformans has evolved a bipolar mating system  in which the MAT locus is unusually large (>100 kb) and encodes >20 genes including homeodomain (HD) and pheromone/receptor (P/R) genes. To understand how this unique bipolar mating system evolved, we investigated MAT in the closely related species Tsuchiyaea wingfieldii and Cryptococcus amylolentus and discovered two physically unlinked loci encoding the HD and P/R genes. Interestingly, the HD (B) locus sex-specific region is restricted (∼2 kb) and encodes two linked and divergently oriented homeodomain genes in contrast to the solo HD genes  of C. neoformans and Cryptococcus gattii. The P/R (A) locus contains the pheromone and pheromone receptor genes but has expanded considerably compared to other outgroup species (Cryptococcus heveanensis) and is linked to many of the genes also found in the MAT locus of the pathogenic Cryptococcus species. Our discovery of a heterothallic sexual cycle for C. amylolentus allowed us to establish the biological roles of the sex-determining regions. Matings between two strains of opposite mating-types (A1B1×A2B2) produced dikaryotic hyphae with fused clamp connections, basidia, and basidiospores. Genotyping progeny using markers linked and unlinked to MAT revealed that meiosis and uniparental mitochondrial inheritance occur during the sexual cycle of C. amylolentus. The sexual cycle is tetrapolar and produces fertile progeny of four mating-types , but a high proportion of progeny are infertile, and fertility is biased towards one parental mating-type (A1B1). Our studies reveal insights into the plasticity and transitions in both mechanisms of sex determination (bipolar versus tetrapolar) and sexual reproduction (outcrossing versus inbreeding) with implications for similar evolutionary transitions and processes in fungi, plants, and animals.Sexual reproduction in fungi is governed by a specialized genomic region called the mating-type locus ( C. neoformans/C. gattii mating-type (MAT) locus spans a single >100 kb gene cluster encoding >20 genes, many involved in sex. We examined MAT gene cluster evolution in model and pathogenic Cryptococcus species. MAT was characterized from two closely related species, T. wingfieldii and C. amylolentus, and is organized into two unlinked gene clusters on different chromosomes. MAT organization in these species provides insight into evolutionary transitions from tetrapolar to bipolar mating systems involving fusion of physically unlinked sex-determinants into one contiguous region. These sex determination transitions occurred concomitantly with the origin of the pathogenic species complex from the last common ancestor shared with tetrapolar non-pathogenic species. We discovered a tetrapolar sexual cycle in C. amylolentus that generates recombinant meiotic progeny, many of which are infertile. Fertile progeny are biased towards one parental mating-type (A1B1) and may be an evolutionary precursor to unisexual mating of the closely related pathogenic species. This study reveals factors orchestrating gene cluster formation and sex chromosome evolution in fungi, including features shared with animals and plants.Fungal gene clusters mediate sex determination, natural product synthesis, and metabolic functions. Eukaryotic organisms share features of gene cluster formation including translocations, inversions, gene conversion, and suppressed recombination. The  Sexual reproduction is ubiquitous throughout nature, generates population diversity, and has been described extensively in plants, animals, and microorganisms MAT) are encoded by a single locus; in some examples genes encoding pheromones and their receptors are also present MAT (a and α), although there are examples of bipolar fungi that also undergo same-sex mating  control and establish cell identity. These loci are often multiallelic, and alleles must differ at both loci for sexual reproduction to occur. Bipolar mating systems support more efficient inbreeding (50%) and also outbreeding (50%), while tetrapolar systems promote more efficient outbreeding (>99%) and restrict inbreeding (25%) Saccharomyces cerevisiae and Candida albicans are bipolar while basidiomycetous yeasts like Tremella mesenterica and Ustilago maydis are typically tetrapolar Ustilago hordei, Coprinellus disseminatus, C. neoformans, and Cryptococcus gattii have bipolar mating systems Fungi occur in two mating configurations: bipolar and tetrapolar C. neoformans is a haploid, dimorphic fungus that has a bipolar mating system, represented by two alleles, α and aMAT spans 100 to 120 kb, and encodes more than 20 genes, many of which are involved in mating. Comparison of the MAT gene cluster among the members of the pathogenic Cryptococcus species complex revealed that extensive rearrangements and gene conversions have occurred over time even though recombination in this gene cluster is generally suppressed MAT in the pathogenic Cryptococcus species have been extensively examined and are well defined Cryptococcus reproduces via either opposite-sex or unisexual reproduction a) initiates with cell-cell fusion, followed by production of a filamentous dikaryon with fused clamp cell connections, and culminates in nuclear fusion and meiosis in the basidia 1-α2) or two genetically identical genomes (α1-α1), and the resulting meiotic spore products are all α.MAT to the pathogenic Cryptococcus species was tetrapolar, with the homeodomain (HD) and pheromone/receptor (P/R) genes present in two unlinked sex-determining regions MAT loci. In this model, a chromosomal translocation event then fused the unlinked loci into a contiguous region resulting in the formation of a transient tripolar intermediate in which MAT is linked in one partner yet unlinked in the other. This unstable intermediate underwent gene conversion to link the other MAT locus alleles, one or the other homeodomain gene was lost, and MAT was subjected to multiple inversions and gene conversions events to yield the extant bipolar MAT locus of CryptococcusFraser et al. proposed that the ancestral form of Cryptococcus species form a monophyletic cluster composed of at least two but possibly as many as six species: C. neoformans var. neoformans, C. neoformans var. grubii, and the sibling species C. gattii  that all have the potential to infect humans and other animals sensu stricto Filobasidiella clade is comprised of the pathogenic species and three closely related saprobic species: Tsuchiyaea wingfieldii, Cryptococcus amylolentus, and Filobasidiella depauperatasensu lato sister clade Kwoniella encompasses several saprobic and one aquatic-associated species: Bullera dendrophila, Cryptococcus heveanensis, Cryptococcus bestiolae, Cryptococcus dejecticola, and Kwoniella mangroviensisThe pathogenic Cryptococcus species complex, sex has recently been described for C. heveanensis and K. mangroviensisKwoniella clade and basidiospores associated with cruciate-septated basidia are produced during mating. Additionally in F. depauperata and T. mesenterica, the nature of sex has also been revealed in previous studies and exemplifies homothallic and heterothallic sexual cycles, respectively F. depauperata resemble the basidia and basidiospores of C. neoformans and C. gattii while T. mesenterica mating products are similar to C. heveanensis and K. mangroviensisC. amylolentus or T. wingfieldii.Of these species that are phylogenetically closely related to the pathogenic C. heveanensis revealed it has a tetrapolar mating system, i.e. its sexual reproduction is governed by MAT comprised of two physically unlinked gene clusters: a multiallelic HD locus (B locus) and a P/R locus (A locus) that is at least biallelic Cryptococcus pathogenic species first appeared, that is, did it emerge earlier in the common ancestor of the sensu stricto group when it split from the sensu lato group, or did it evolve later and only in the Cryptococcus pathogenic species? Given the close relationship of T. wingfieldii and C. amylolentus to the pathogenic Cryptococcus species complex, understanding their life cycles, as well as their MAT loci configurations can provide key insights into the evolution of MAT and sexual reproduction in C. neoformans and C. gattii.A recent study of C. amylolentus that we observed under laboratory conditions. Additionally, we characterized the MAT loci of T. wingfieldii and C. amylolentus, and discovered in both species two physically unlinked gene clusters, one encoding the HD locus and the other encoding the P/R locus. Genes within these clusters include many homologs of Cryptococcus MAT-associated genes. Furthermore, our mating assay and genetic analyses of C. amylolentus meiotic progeny showed that many meiotic progeny are sterile and one parental type is overrepresented in the meiotic products, suggesting its tetrapolar mating system deviates from the classic model. We discuss the implications of our findings in the context of the evolution of the mating type locus as well as of bipolar sexuality in the Cryptococcus species complex. Our findings also provide insights into similar evolutionary processes that drive the formation and function of sex chromosomes in algae, fish, insects, and mammals In this study, we provide a detailed description of the heterothallic sexual cycle of MAT in T. wingfieldii, fosmid libraries were constructed from the type strain CBS7118 and probed with several genes within  or flanking (FAO1 and NOG2) the C. neoformans MAT locus. Positive clones (3F11-3A15-5J15 (P/R locus), 2B23-2K10 (HD locus), and 4E07 (FAO1), see MAT loci. The FAO1 gene lies on a distinct fosmid and appears to be unlinked or distant from MAT. The region obtained containing the P/R locus spans ∼70 kb and the region obtained containing the HD locus spans ∼40 kb  employing the same approach. Fosmid libraries were generated and probed with MYO2, LPD1, RPL39, and SXI1. Primers specific for MAT genes in T. wingfieldii were used to generate probes for C. amylolentus and the identity of each probe was confirmed via cloning and sequencing. Positive clones (4E01 (SXI1), 4E22 (MYO2), and 3H19 (LPD1), see MAT loci. An additional fosmid (3N14 (RPL39), see C. amylolentus. The gene content in MAT appears to be largely conserved between T. wingfieldii and C. amylolentus. However, our Southern blot analysis indicated at least two major inversions exist between the P/R regions of these two species . In T. wingfieldii, three pheromone genes  are present and share greater identity with the MFa genes of C. gattii with an identity of 80% compared to 70–75% shared with the MFα pheromone gene  have been identified and share 73% identity with the MFa protein product of C. neoformans and 65–70% identity with the MFα pheromone gene. In summary, both SXI1 and SXI2 were present in the ancestral HD locus of the sensu stricto Filobasidiella clade. Thus, loss of one or the other HD gene occurred during the evolution of MAT in the pathogenic Cryptococcus species. Additionally, the five genes most recently acquired by the Cryptococcus MAT locus are linked to the ancestral P/R locus and thus appear to have been acquired into the expanding MAT A locus rather than entrapped by the MAT fusion event, in contrast to an earlier evolutionary model, suggesting a revision to the model . In the MAT locus of the pathogenic Cryptococcus species, STE11 is present. In C. heveanensis, STE11 is linked to but distant from the P/R locus and this may represent the ancestral configuration with retention in C. neoformans and C. gattii and translocation out of MAT in C. amylolentus and T. wingfieldiiT. wingfieldii, the flanking gene at the 3′ end of MAT, NOG2, was used as a probe. It was present in a single contig within the larger fosmid assembly of T. wingfieldii, but has not been linked to either the HD or P/R loci contigs. PCR analysis (using gap closure) revealed that LPD1 is linked to the P/R locus, although this gap remains to be sequenced. Interestingly, NCP1 and NCP2 are duplicated genes in T. wingfieldii and C. amylolentus but not in the pathogenic Cryptococcus species. The NCP1/2 genes are also duplicated in C. heveanensisIn C. neoformans genes as the most closely related homolog in other sequenced fungal genomes. Four of these genes reside on chromosome 4 and two on chromosome 5 of C. neoformans, indicating that translocation  events may have occurred between these two chromosomes during the evolution of MAT in the pathogenic Cryptococcus species C. heveanensis, F. depauperata, and T. mesenterica there is additional evidence for similar exchanges between chromosomes We also identified several hypothetical genes  with MAT loci in T. wingfieldii and C. amylolentus, but we also observed at least two major inversion events that have occurred between the two genomes  have expanded but not yet fused.A considerable level of synteny exists across both hout MAT , similarT. wingfieldii, as well as in both strains of C. amylolentus (CBS6039 and CBS6273). Each genome has approximately 10–12 chromosomes ranging in size from 800 kb to 2.2 Mb. Three genes were used to probe the T. wingfieldii chromosomes, two from the HD locus, SXI1 and RPL22, and one from the P/R locus, MYO2  are also tetrapolar MAT and the bipolar state observed for the pathogenic Cryptococcus species therefore arose even more recently than revealed by previous studies of the more distantly related sensu lato species C. heveanensisMAT in the sibling species resembles key aspects of the proposed intermediates in the evolution of bipolar MAT in the pathogenic Cryptococcus species from a tetrapolar ancestor.Analysis using pulsed-field gel electrophoresis and Southern hybridization demonstrated that the HD and P/R loci are physically unlinked in us, MYO2 . For C. and ETF1 . From thMAT is defined as a gene cluster (containing either HD and/or P/R genes) whose sequence is divergent between two strains of opposite mating-types. Based on the characterized structure of MAT in both species, we sought to determine which genes in each region govern and control sexual identity. The lack of additional T. wingfieldii strains has made it difficult to assess experimentally whether it has a sexual cycle and, if so, which genes are involved. Fortunately, in C. amylolentus two strains are available and this enabled our analysis of MAT and sex in this species resulting in the discovery of an extant sexual cycle (described below).MAT typically display polymorphisms when comparing sequences from strains of opposite mating-type while the genes that flank MAT share a much higher level of identity (≥99%). The SXI1 and SXI2 dimorphic region defines the diverged region of the MAT B HD locus in C. amylolentus. We aligned the nucleotide sequences and performed a matrix comparison for the dimorphic region (∼2 kb) spanning the SXI1 and SXI2 genes in CBS6039 and CBS6273. The diversity lies in the region between the two genes, and their divergently oriented 5′ regions span roughly 600 bp with a similarity score of 92% , our analysis based on PCR assay showed that the areas flanking the SXI1 and SXI2 genes are conserved enough between CBS6039 and CBS6273 that primers designed based on CBS6039 sequence amplify corresponding regions from CBS6273 (data not shown).Regions that define e of 92% . This reSTE3 lies within the A P/R mating-type locus, we performed Southern blot analysis using genomic DNA from the two strains of C. amylolentus. The STE3 PCR product derived from CBS6039 was used as a probe, and only hybridized to the lanes containing CBS6039 DNA with no hybridization to CBS6273 . This contrasts with C. heveanensis in which the P/R locus is more restricted, STE3 and the MF pheromone genes are closely linked, and the LPD1, STE11, ZNF1, MYO2, and IKS1 genes are linked to but not within MATC. amylolentus a tetrapolar mating system with physically unlinked HD and P/R loci appears to define mating-type identity, and the P/R locus has expanded considerably compared to C. heveanensis, revealing an evolutionary intermediate in the transition from the tetrapolar to bipolar state that is even more closely related to the pathogenic species complex.To determine whether the pheromone receptor gene  CBS6273 . This anMAT locus of C. neoformans . This analysis included C. neoformans var. neoformans, C. neoformans var. grubii, and C. gattii representatives from the pathogenic species cluster C. amylolentus and T. wingfieldii, as well as the outgroup species C. heveanensis and T. mesentericaC. neoformans pathogenic species cluster, these genes can be classified into three different groups: species specific , mating-type specific , and mating-type unique genes    and S7.CID1, GEF1, and LPD1 is consistent with the hypothesis that this region has been recruited into the MAT locus of C. neoformans during the transition from a tetrapolar to a bipolar mating system. For the sex-unique genes in the C. neoformans species complex, SXI1 and SXI2, SXI2 showed a considerably higher level of polymorphism between the two alleles from the CBS6039 and CBS6273 C. amylolentus isolates  spores per chain and clamp cell connections are visible by light microscopy and SEM  and monokaryotic  hyphae while F. depauperata produces only monokaryotic hyphae. The presence of dikaryotic hyphae in C. amylolentus provides evidence that opposite-sex mating occurs during the sexual cycle In  and SEM . Based o and SEM –S8D. In Cryptococcus species. In summary, microscopic examination of mating structures in C. amylolentus has revealed both shared hallmarks with sexual reproduction in the pathogenic Cryptococcus species and novel features.Interestingly, the cap of the spore chain represents a quartet of basidiospores. These spores are the oldest in the spore chain and remain tightly attached to each other. Younger spores in the four spore chains remain attached to the preceding and following spores in the chain but often not to their meiotic siblings in the other three spore chains. Thus, the quartet spore cap appears to tether the ends of the spore chains together. This feature has not been described in the pathogenic C. amylolentus is tetrapolar or bipolar, we performed microdissection of random progeny (F1 set 1) and individual spore chains (F1 set 2) followed by molecular genotyping analysis for both MAT markers and a genome-wide set of RAPD markers. We designate the CBS6039 parent as A1B1 and the CBS6273 parent as A2B2, according to the designation used for a tetrapolar mating system and our findings, assigning A as the P/R locus and B as the HD locus . Genotyping using MAT markers and RAPD markers revealed that most of the progeny inherited all of the parental alleles from CBS6039 (A1B1)  that revealed a majority of this progeny set possess nuclear and mitochondrial genomes inherited from different parents. Remarkably, 22 (78%) of the progeny are sterile and unable to undergo sexual reproduction with either parent or their F1 siblings. It is interesting that progeny that appear to be derived from blastospores are, for unknown reasons, frequently sterile.For F1 set 1 (F1S1), a total of 40 spores were dissected and 28 (70%) germinated  and S2. 9 (A1B1)  and S2 aMAT A or B genes revealed that 64.5% (20/31) of the progeny resembled one or the other parent (A1B1 or A2B2) while the other ∼35% exhibit evidence of recombination within the P/R locus and/or between the HD and P/R loci (i.e. A1B2 or A2B1 progeny)  from four well-resolved individual spore chains (one chain each from four different basidia). The germination frequency was 91% (31/34), and 58% (18/31) of the progeny were sterile with both parents . All of progeny) . In contprogeny) . Linkageprogeny) . SpecifiMAT recombinant progeny. Specifically, successful mating was observed when A1B2 progeny (F1S2 #10 and #16) and A2B1 progeny  were co-cultured together (with the exception of mating between F1S2 #16 and F1S1 #18). None of these MAT recombinant progeny mated with either parent, further confirming that C. amylolentus possesses a tetrapolar mating system , there are 14 A1B1 F1 progeny that are fertile with the A2B2 parent CBS6273, one A2B2 progeny that is fertile with the A1B1 parent CBS6039, and one that is fertile with both parents. Among the spore chain derived (F1S2), progeny #13 (A2B2) was found to be able to mate with progeny #3 and #24 (A1B1). Successful mating was also observed between some g system .Because only 32% (19/59) of the progeny are fertile in both progeny sets, we assessed whether fertility increases with an additional sexual cross or mitotic passage. Even after several passages on YPD, the sterile phenotype remained stable (data not shown). We crossed F1S2 progeny #3 and CBS6273 to generate a backcross progeny set (F2). Interestingly, most of the basidia in the cross were barren and if spore chains were present, the number of spores per chain was significantly reduced when compared to matings between the parental strains. We were successful in dissecting spores from two individual spore chains. The germination rate was 54% (6/11) and all of the progeny were fertile (50% with the CBS6039 parent and the remaining A2B1 progeny are interfertile with the F1S2 progeny #10 and #16 ). All ofC. amylolentus conforms to a modified tetrapolar mating system in that 1) sterile progeny are also frequently produced, and 2) the ratio of the four mating types is unbalanced.In summary, taken together our genotyping data indicates that meiotic recombinants are present among the sexually produced progeny and our evidence is that the sexual cycle of C. amylolentus, SNPs were first identified in two mitochondrial genes, NAD4 and NAD5, between the two parental strains, CBS6039 and CBS6273, by PCR amplification and sequencing. Of the 65 progeny screened, no intra- or inter-genic recombination between the two genes was observed, and all of the progeny (with the exception of two from F1S1) typed as the CBS6273 (A2B2) parent  parent . The twoMAT locus in the pathogenic Cryptococcus species to the closest known species, T. wingfieldii and C. amylolentus. To determine the structure of MAT in both species, we cloned and sequenced the HD and P/R loci. Due to their close phylogenetic relatedness MAT has provided key insights into the evolution of MAT and revealed important aspects of the transition from an ancestral tetrapolar to a bipolar mating system in C. neoformans and C. gattiiThe current study extends the previous analyses of the Cryptococcus species complex sensu stricto (closely related) and sensu lato (more distantly related) species that provide unique vantage points to address questions such as: when and how did the bipolar mating system evolve? And what, if any, affects does the emergence of bipolar mating systems have on the pathogenesis of C. neoformans and C. gattii? Previous studies on a more distantly related sensu lato species, Cryptococcus heveanensis, revealed it to be tetrapolar sensu stricto strains, C. amylolentus and T. wingfieldii are much more closely related to the pathogenic species C. neoformans/C. gattii than is C. heveanensis; hence the transition to bipolarity in the pathogens was even more recent than could be concluded based on the studies of C. heveanensis alone. Second, by providing additional tetrapolar outgroup species, we can conclude that the transition was from tetrapolar to bipolar, not vice versa. Third, the P/R locus is much more expanded in C. amylolentus compared to C. heveanensis, providing further insights on the evolution of the MAT and this key step in the process. Furthermore, the tetrapolar mating system in C. amylolentus showed indications of deviation from the classic tetrapolar model in that many MAT loci recombinant progeny are sterile and progeny that resemble one parent at the MAT loci dominate the progeny population. Moreover, the organization of MAT in these sibling species mirrors key aspects , which shaped the evolution of the mating-type locus in the pathogenic Cryptococcus species complex.A previous phylogenetic analysis using a six-gene multi-locus sequencing (MLS) approach identified the most closely related species to the pathogenic Cryptococcus species cluster and revealed that T. wingfieldii and C. amylolentus are sibling species, the closest relatives of the pathogenic species, and members of the Filobasidiella clade MAT loci of T. wingfieldii and C. amylolentus share overall synteny, with two major inversion events present between the P/R loci of the two species  are present and these lie within MAT in C. neoformans but appear to lie outside of MAT in C. amylolentus. The configuration of the HD genes in the sibling species provides evidence that the ancestral form of the HD locus contained both SXI1 and SXI2, similar to tetrapolar mating systems in other basidiomycetes, and that loss of one or the other of the HD genes punctuated the formation of a bipolar mating system.Previous analysis resolved the phylogeny surrounding the pathogenic  species . For thiomosomes . The MATB) and P/R (A) loci of the sibling species, we determined which genes define MAT. Because only one strain of T. wingfieldii is available, we were unable to establish which of the genes in the B and the A loci are MAT-specific. By comparing sequences from the two strains of C. amylolentus, we determined that the MAT-specific region in the HD locus is likely restricted to the ∼3 kb SXI1 and SXI2 dimorphic region. The divergence is present in the 5′ regions of SXI1 and SXI2, similar to recent findings on the B MAT locus alleles of C. heveanensisOf the >20 genes identified in the HD  causing sterility in the C. amylolentus progeny is not clear, there are several possible explanations. First, it is possible that aneuploids (1N+1) are generated during meiosis that could be sterile. FACS analysis of the examined progeny suggested that all of the progeny are haploid with the exception of a single diploid (F2 progeny #4), but FACS is not sensitive enough to detect 1N+1 aneuploids. Employing comparative genomic hybridization (CGH) of the C. amylolentus parental strains with the sterile progeny will be necessary to address the issue of possible aneuploidy generated during mating. Second, meiosis is mutagenic and sexual reproduction may also increase transposition in the genome. The resulted mutations and/or the insertion of transposons in MAT or elsewhere might result in sterility. Third, the increased sterility among progeny could be due to sex induced silencing of repetitive elements within MAT and linked fertility genes MAT caused by gene conversion events. Sex induced silencing requires the RNAi machinery. However it is not known yet if C. amylolentus possesses these genes. The C. amylolentus genome sequence will allow this question to be answered. Additionally, we cannot exclude the possibility that the sterility observed among the progeny is due to divergence/incompatibility between the mating machineries of the two C. amylolentus strains, or to nuclear-mitochondrial incompatibility that has been observed in other yeasts Mating in C. neoformans, meiosis typically gives rise to four meiotic products and it was recently shown that a single meiotic event occurs in each basidium C. amylolentus, more than one meiotic event could occur in the basidium. However, this would have to involve post meiotic nuclear fusion and a second round of meiosis. In this case, up to eight genotypes could be produced from one basidium. Also, high gene conversion events favoring some alleles over others could result in a non-Mendelian inheritance pattern and skew the resulting genotypes in each individual spore chain.From the genotyping analysis, it is evident that extensive recombination occurred among the progeny produced by sexual reproduction. Additionally, we observed a 1∶1 segregation pattern of the two parental alleles in the progeny population. This segregation data and the high level of genetic exchange in the progeny  provide strong evidence that meiosis occurs within the basidium during sexual reproduction. Additionally, RAPD analysis revealed that in some spore chains from the F1S2 and the F2 progeny sets, more than four genotypes are present in a single chain. There are several possible explanations. In Another possible explanation for the observed >4 genotypes/basidium that we favor is the presence of aneuploids in the progeny population. The RAPD markers employed in this analysis differentiate the two parental strains by the presence or absence of a PCR product. If progeny are aneuploid for one or more chromosome, they could appear unique and differ from the two parental strains. In this aneuploidy model we expect the basidiospores from one basidium to share four common genotypes with the exception of a few rarer genotypes . This model is consistent with our RAPD data  in whichMAT loci (A1B2 and A2B1), and these MAT recombinant progeny are inter-fertile, but cannot mate with either of the two parental strains, proving that C. amylolentus has a tetrapolar mating system. However, among those isolates that were fertile the A1B1 genotype was overrepresented, whereas the other three genotypes were underrepresented  that might have ultimately led to the emergence of unisexual same-sex mating in C. neoformans. In conclusion, C. amylolentus and C. heveanensis have physically unlinked HD and P/R loci and this arrangement further supports tetrapolarity as the ancestral configuration, and that the transition to bipolarity occurred recently and concomitantly with the emergence of the pathogenic C. neoformans/C. gattii species cluster.Results from our studies illustrate features of both the transition from tetrapolarity to bipolarity in the closely aligned saprobic and pathogenic These studies on the molecular events leading to the fusion of two unlinked sex determining regions of the genome in the ancestral tetrapolar state to the derived bipolar mating systems mirror aspects in the hypothesized origin of sex chromosomes of more complex multicellular eukaryotes, including plants, insects, fish, and mammals. Namely, Ohno hypothesized that sex determinants arise on an autosome, and then gradually capture this chromosome, which evolves to become a sex chromosome C. amylolentus, CBS6039 and CBS6273, and the one T. wingfieldii isolate, CBS7118, were obtained from the Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre in the Netherlands. Both CBS6039 and CBS6273 were originally isolated from insect frass in South Africa, while CBS7118 was originally isolated from rubber sheet in Indonesia. All species were grown and maintained on yeast extract-peptone-dextrose (YPD) medium at 24°C. Mating assays were performed on V8 medium pH = 5 in the dark and also at 24°C. Random spore dissection was performed on YPD medium as previously described The two strains of T. wingfieldii and C. amylolentus, cells were cultured in 50 ml of liquid YPD shaking overnight at 24°C. The pellets were then lyophilized overnight and the CTAB method of fungal DNA isolation was performed as described before To isolate genomic DNA from http://blocks.fhcrc.org/codehop.html) to identify MAT specific genes in T. wingfieldii. The primers consist of a relatively short 3′ degenerate core and a longer 5′ non-degenerate consensus clamp designed by multiple sequence alignments FAO1 and NOG2) and two recently acquired MAT genes (RPO41 and LPD1) from C. neoformans var. neoformans and var. grubii, C. gattii, U. maydis, and C. cinerea to design the degenerate PCR primers . Plasmid DNA was purified from transformants and then sequenced. For C. amylolentus, degenerate primers were not used. Instead, primers from T. wingfieldii were directly used to amplify MYO2, LPD1, SXI1, and SXI2 in both C. amylolentus strains  on a CHEF DR-II apparatus . The following conditions were used: 1- to 6-second switch time, 6 V/cm, 14°C for 14–15 hrs in 0.5X TBE. The size-fractionated DNA, 25 to 40 kb fragments, was recovered by gel extraction and the DNA was precipitated with sodium acetate and ethanol. The precipitated insert DNA was then ligated into the CopyControl pCC1FOS cloning-ready vector and incubated overnight at 24°C. The ligated DNA was packaged in phage particles and plated on E. coli phage-resistant cells (EPI100-T1R plating strain) overnight at 37°C (detailed protocol can found at http://www.epibio.com/item.asp?ID=385). Approximately 16,000 fosmid clones were picked into 96-well plates and transferred to 384-well plates for long-term storage at −80°C. The 384-well plates were replicated onto high-density filters for hybridizations using the MAT genes.We employed the CopyControl Fosmid Library Production Kit  to generate fosmid libraries for T. wingfieldii and four fosmids were individually sequenced to generate the assembly for C. amylolentus strain CBS6039. Sequencing reactions were performed using Big Dye chemistry v3.1  and analyzed on an Applied Biosystems 3730xl capillary sequencer in the Biological Sciences Sequencing Facility at Duke University. For each library, approximately ∼1200 sequence reads were imported into UNIX using Phred and Phrap to assemble the sequences into larger contigs of overlapping sequence http://frodo.wi.mit.edu/primer3/). The GenBank accession numbers for T. wingfieldii are HM368525 (HD locus) and HM368524 (P/R locus). The GenBank accession numbers for the HD locus and the three P/R contigs in C. amylolentus CBS6039 are: HM640220 (HD locus), HM640221 (RPL39-MYO2), HM640222 (LPD1-STE12), and HM640223 (GEF1-MFA). The GenBank accession numbers for genes from C. amylolentus CBS6273 are: HM640224 (SXI1), HM640225 (SXI2), HM640226 (GEF1), HM640227 (LPD1), and HM640228 (ETF1).Positive fosmid clones were sequenced using the shot-gun sequencing method described by Metin et al. C. amylolentus and one T. wingfieldii strains, we cultured the isolates on YPD medium for 2 days at 24°C. Each isolate was processed for flow cytometry as previously described C. neoformans/gattii was used as a reference for ploidy determination (including haploid and diploid controls).To determine the ploidy of the two C. amylolentus and T. wingfieldii, spheroplasts were generated following the spheroplasting protocol for C. neoformans and C. gattiiMAT gene probes generated by PCR. We also performed Southern blot analysis on genomic DNA from C. amylolentus that was digested with EcoRV, PstI, BamHI, or NotI. The digested DNA was separated on an agarose gel and probed with the RPL22 gene probe amplified from C. amylolentus, with primers designed for T. wingfieldii  analysis. To generate each dot plot, we employed the Molecular Toolkit's online nucleic acid dot plots program (http://www.vivo.colostate.edu/molkit/dnadot/). The parameters for the dot plot analyses were as follows: the window size was 51 and the mismatch limit was 6. We also employed the bioinformatic software, Artemis Comparison Tool Release 8 (http://www.sanger.ac.uk/resources/software/) to generate comparison plots across MAT of T. wingfieldii to C. amylolentus and both sibling species compared to C. neoformans serotype D strain JEC21 http://www.webact.org/WebACT/home) with the Blastn algorithm We compared sequences from the HD locus of Phylogenetic analysis was performed on coding sequences using MEGA 5 C. amylolentus digested with BamHI, BglI, ClaI, EcoRI, or NcoI. The digested DNA was separated on an agarose gel and probed with the STE3 gene, stripped , and probed with the contig ends from the P/R assembly in C. amylolentus amplified by PCR  Golubev 1981 Etymology: The epithet is chosen to be identical with that of C. amylolentus type strain, CBS6039T (A1B1) crossed to CBS6273 (A2B2) on V8 medium (pH = 5). These strains were originally isolated from insect frass in South Africa Filobasidiella amylolenta.Holotype: Mounted teleomorph is paired cultures of Filobasidiella amylolenta Findley & Heitman sp. nov.Latin description: Fungus heterothallicus. Hyphae dikaryoticae, fibulis fusis. Basidia aseptata, 3–5 µm lata, quatuor catenas basidiosporarum producentia. Basidiosporae aeriae, globosae, 2–2.5 µm diametro.C. amylolentus matings incubated on V8 pH = 5 medium for 2 weeks. The specimen was prepared and analyzed as described in Spores and yeast cells were cultured on slides coated with V8 pH = 5 medium for one week or longer to allow production of mating structures. The slide was first washed with phosphate buffered saline (PBS) followed by staining the cell wall using a solution of Calcofluor white  for 15 minutes. Slides were rinsed with PBS and fixed for 15 minutes in fixing solution . After permeabilization of the fungal cells, nuclear content was examined by staining with Sytox green (Molecular Probes) for 30 minutes. Slides were washed with PBS and a cover slip was applied to the slide for observation. In addition to staining spores and yeasts, mating filaments were also stained. Agar pieces were removed from mating plates and washed several times with PBS. Calcofluor white was added directly to the agar piece for 30 minutes, followed by washing with PBS, and fixing for 45 minutes. After permeabilizing samples, filaments were washed with PBS and stained with 1 mg/ml Hoechst 33258  overnight at 4°C. The next day, samples were washed with PBS, a thin slice of the agar (containing the mating filaments) was removed using a razor blade and a mounting solution containing anti-fade  was added to the agar slice on a slide. The slides were sealed with nail polish and stored at 4°C in the dark after microscopic evaluation. All staining was performed at 24°C, unless otherwise noted. SEM was performed on Microdissection of spores ) was performed on YPD medium incubated at 24°C for two days to allow spores to germinate.MAT loci was achieved using a set of PCR markers  and PCR-RFLP markers (SXI1 (enzyme EcoRV), SXI2 (enzyme RsaI), and ETF1 (enzyme DdeI)). To genotype other genomic regions, we used a set of 20 RAPD markers . We designate the CBS6039 parent as A1B1 and the CBS6273 parent as A2B2, according to the designation used for a tetrapolar mating system and our findings, assigning A as the P/R locus and B as the HD locus  constituting the HD locus, three overlapping fosmids  constituting the P/R locus, and a separate fosmid (4E07) containing the gene FAO1 and unlinked to either the HD or P/R locus, were sequenced. The MAT loci are embedded within regions spanning a total ∼110 kb.Fosmid map of the HD and P/R assembly in (TIF)Click here for additional data file.Figure S2C. amylolentus. One fosmid (4E01) constitutes the HD locus while three fosmids  span the P/R locus. The regions containing the MAT loci represent ∼80 kb in total. Southern blots are shown in the bottom half of the figure. This data supports the current assembly of the C. amylolentus P/R locus shown above. In blots (a), (b), and (e), genomic DNAs from CBS6039 and CBS6273 were digested with BamHI, while for blots (c), (d), and (f) DNA was digested with ClaI (left) or EcoRI (right). The probes used for Southern blot analysis were located at the ends of the three contigs (indicated with the red block arrows). Sizes of the restriction fragments are also indicated. For probes (a), (b), (c), and (e), no hybridization signals were detected for CBS6273, indicating high levels of nucleotide polymorphism between CBS6039 and CBS6273 at these regions. Probe labels 1L, 1R, 2R, 3L, and 3R correspond to those in Fosmid map of the HD and P/R assembly in (TIF)Click here for additional data file.Figure S3Estimations of the sizes of the gaps within the PR locus assembly based on Southern blotting. The numbers in the parentheses indicate the locations of the restriction enzyme recognition sites within their respective contigs. Block arrows show the locations of the probes used for Southern blotting (see images in (TIF)Click here for additional data file.Figure S4Results of PCR assay indicating similarity and divergence between CBS6039 and CBS6273 within the P/R locus assembly. Primers were designed based on the CBS6039 P/R locus assembly, and were used for PCR reactions using either CBS6039 or CBS6273 genomic DNA as template. Black squares indicate primer pairs that produced PCR products in both strains; gray squares indicate primer pairs that yielded PCR products for CBS6039 but not for CBS6273; white squares indicate primer pairs that yielded no PCR product for either strain. Genes from the CBS6039 P/R locus assembly are labeled at the top. Gaps No.1 and No.2, as well as the contig end labels  correspond to those in (TIF)Click here for additional data file.Figure S5MAT in the sibling species and C. neoformans strain JEC21. MAT sequences from T. wingfieldii and C. amylolentus were compared to C. neoformans and a synteny analysis was performed. Red denotes conserved gene order while blue indicates inversion events. Green bars under the assembly denote gaps in assembly.Extensive chromosomal rearrangements are present throughout (TIF)Click here for additional data file.Figure S6C. amylolentus genes. The phylogenetic relationship of C. amylolentus and T. wingfieldii to the pathogenic Cryptococcus species and neighboring taxa is highlighted and four representative genes GEF1, CID1, STE12, and ETF1 are shown. GEF1 and CID1 exhibit a species-specific phylogeny in C. neoformans and C. gattii, while STE12 and ETF1 exhibit mating-type specific phylogeny. The trees were constructed using the Neighbor-Joining method implemented in the software MEGA4. Bootstrap values on tree branches were calculated from 500 replicates. (α) indicates strains with the MATα locus, and (a) indicates strains with the MATa locus.Phylogenetic analysis of additional (TIF)Click here for additional data file.Figure S7SXI1 and SXI2 in C. amylolentus. The SXI1 and SXI2 genes were analyzed in C. amylolentus and neighboring taxa and the gene trees were constructed using the Neighbor-Joining method implemented with the software program MEGA4. Bootstrap values on tree branches were calculated from 500 replicates. (α) indicates strains with the MATα locus, and (a) indicates strains with the MATa locus.Phylogenetic analysis of the homeodomain transcription factor genes, (TIF)Click here for additional data file.Figure S8C. amylolentus mating structures. (A and B) Staining of basidiospores, scale bars = 2 µm. (A) Differential Interference Contrast (DIC) image, and (B) fluorescence image of basidiospores nuclei stained with Hoechst 33258. (C and D) Staining of a dikaryotic mating filament. (C) DIC image, and (D) fluorescence image of mating filaments, in which nuclei were stained with Hoechst 33258. (E–F) Staining of mating filament and clamp cell. (E) Nuclear content of filament stained with Sytox green and (F) Calcofluor white for cell wall visualization. (G) Nuclear content of basidiospores stained with Sytox green and (H) cell wall with Calcofluor white .Fluorescence microscopy of (TIF)Click here for additional data file.Figure S9Evidence for one meiotic event in each basidium. Three basidia were analyzed: basidium A includes F1S2 progeny 1 to 7; basidium B includes F1S2 progeny 8 to 17; basidium C includes F1S2 progeny 18 to 24. The genetic distances were calculated using the UPGMA method implemented with the MEGA 5 program. For all three basidia, each harbors four major clusters of consensus progeny genotypes. Some clusters have several progeny that are closely related yet slightly differ from a majority genotype. This is consistent with a scenario in which one meiosis event occurred during sexual reproduction, and some atypical progeny are aneuploid at a few genetic loci from the consensus meiotic genotypes.(TIF)Click here for additional data file.Figure S10SXI1 and SXI2 dimorphic region is similar in CBS6039 and F1 set 2 progeny #4. Percent identity plots comparing the ∼2 kb region containing the HD genes in the C. amylolentus parental strain CBS6039 compared to set 2 F1 progeny #4.The (TIF)Click here for additional data file.Figure S11SXI1 and as CBS6273 at SXI2. RFLP analysis of the recombinant F1S2 progeny #16. The progeny and both parental strains were digested with EcoRV and RsaI at SXI1 and SXI2, respectively. This analysis reveals a short gene conversion track or very local double crossover event resulted in a novel B MAT locus marker allele. These enzymes only cleave the PCR product in the parental strain CBS6039. L = 1 kb ladder.F1 Set 2 progeny #16 is recombinant and types as CBS6039 at (TIF)Click here for additional data file.Figure S12SXI1-SXI2 dimorphic region differs between CBS6273 and progeny set 2 F1 progeny #16. Percent identity plots comparing the ∼2 kb region containing the HD genes in the C. amylolentus parental strain CBS6273 compared to set 2 F1 progeny #16. An example of the region of crossover or gene conversion in the HD locus of the recombinant progeny #16 is highlighted with an orange box.The (TIF)Click here for additional data file.Figure S13MAT recombinant progeny and the bi-mater set 1 F1 #4 crossed to the parental strains. Light microscopy of mating structures produced in the interfertile progeny  and backcrosses of progeny #4 to CBS6039 and CBS6273 (bottom row). Scale bar = 10 µm. Mating assays were performed as described in the Mating assays between (TIF)Click here for additional data file.Table S1Random spore dissection of set 1 F1 progeny and molecular analysis of the nuclear markers.(DOCX)Click here for additional data file.Table S2RAPD analysis of F1 set 1 progeny.(DOCX)Click here for additional data file.Table S3List of primers, RAPD markers, and mitochondrial markers used in the study.(DOCX)Click here for additional data file.Table S4Filamentous phenotype and mitochondrial DNA (mtDNA) inheritance identified in all 65 progeny.(DOCX)Click here for additional data file.Text S1RPL22 duplication, and analyses of HD dimorphic region in key progeny.Additional discussions on strains, filamentous phenotype, (DOCX)Click here for additional data file.


Crinivirus includes the whitefly-transmitted members of the family Closteroviridae. Whitefly-transmitted viruses have emerged as a major problem for world agriculture and are responsible for diseases that lead to losses measured in the billions of dollars annually. Criniviruses emerged as a major agricultural threat at the end of the twentieth century with the establishment and naturalization of their whitefly vectors, members of the genera Trialeurodes and Bemisia, in temperate climates around the globe. Several criniviruses cause significant diseases in single infections whereas others remain asymptomatic and only cause disease when found in mixed infections with other viruses. Characterization of the majority of criniviruses has been done in the last 20 years and this article provides a detailed review on the epidemiology of this important group of viruses.The genus  Crinivirus is one of the three genera in the family Closteroviridae and includes viruses with segmented genomes, transmitted by whiteflies (The genus iteflies . DetailsBeet pseudo-yellows virus (BPYV) identified in the 1960s ] encodes up to 10 proteins with a range of functions including but not limited to virus encapsidation, cell-to-cell movement, and vector transmission. Most genomic RNAs have common or highly conserved nucleotides at the 5′ end ranging from 4 to 11 nucleotides in length. The 3′ untranslated regions for each virus other than Lettuce infectious yellows virus (LIYV) share a region of approximately 150 nucleotides with a high degree of genetic conservation between the genomic RNAs.Crinivirus genomic RNAs are encapsidated into long flexuous rods averaging between 650 and 1000 nm in length , and havTrialeurodes and Bemisia in a semi-persistent manner; the reason they are identified with increasing frequency in tropical and subtropical climates where whitefly populations are present. They often cause symptoms that are readily mistaken for physiological or nutritional disorders or pesticide phytotoxicity. Typically, infection is associated with a loss of photosynthetic capability, often characterized by interveinal yellowing of leaves, leaf brittleness, reduced plant vigor, yield reductions, and early senescence, depending on the host plant affected. Some plants may exhibit an interveinal reddening rather than yellowing. Others may exhibit chlorotic mottle on some leaves, usually progressing into interveinal discoloration. Symptoms generally first appear 3–4 weeks after infection, and are most apparent on the older areas of the plant, whereas new growth appears normal. For example, a tomato plant infected with a crinivirus may show extensive interveinal yellowing on leaves near the base, developing interveinal chlorosis on leaves in the middle of the plant, but no symptoms near the apex . Similarly, an infected cucumber plant may appear healthy near the growing point of the vines, but exhibit progressively more severe interveinal yellowing toward the crown . In both cases it is not uncommon for brittle, symptomatic leaves to snap when bent.Crinivirus transmission is species-specific and performed exclusively by whiteflies in the genera An interesting characteristic of many of the criniviruses studied to date is their ability to interact with other viruses in plants and alter symptoms. Studies have shown host-specific competition between crinivirus species that influence accumulation of other viruses present in the plant and consequently symptom severity . Other vBegomovirus, Tomato yellow leaf curl virus (Tomato infectious chlorosis virus (TICV) symptom development in the field by as much as a month compared with controls  as members of each group tend to have similar vectors and host ranges (Table 1).In this communication we provide information on the recent advances in crinivirus epidemiology and associated diseases. Viruses will be presented according to their phylogenetic grouping (Abutilon yellows virus (AYV) is a partially characterized crinivirus originally identified from the common weed velvetleaf (Abutilon theophrasti Medic.) collected from Illinois in 1977  but there is limited information on its host range and its geographic distribution. No crop plants have been identified as hosts; however, the virus can infect members of the Malvaceae, and the experimental solanaceous species, Nicotiana clevelandii A. Grey  is found , reduced growth and fruit size, and early senescence in cucurbits , 99% identical at the amino acid level; Two isolates of BPYV have been fully sequenced, the first from Japan , originaBecause BPYV symptoms are often confused with physiological and nutritional disorders it is likely that the impact and significance of the virus in vegetables and other crops has been underestimated. Additionally, since in most cases symptoms caused by BPYV are those of general plant stress it is important to do virus testing before taking corrective action. Given the great variability among strains, it may be more appropriate to use degenerate primers for virus detection  that wilFigure 3; Tobacco ringspot virus (TRSV) as this was the only virus that was mechanically transmissible from plants with such symptoms. This was questioned when blackberry plants were infected with TRSV using nematodes and infected plants did not develop symptoms over a 3-year period. The first virus characterized from blackberry plants that exhibited BYVD symptoms from South Carolina was a crinivirus, and named Blackberry yellow vein associated virus  is a member of the Rubiaceae (coffee family). Its natural habitat is in wetlands of the Americas, extending between the 45th parallels of both continents. It propagates in a prolific manner through stolons and seed, making it one of the most noxious weeds of turfgrass. Several Virginia buttonweed populations in the southern United States show distinct vein chlorosis or vein netting symptoms, typical of virus infection . T. abutilonea was experimentally verified as a vector. All these properties indicated that Diodia vein chlorosis virus (DVCV) is a member of the genus Crinivirus but no molecular information was available until recently when an isolate from a clone of a plant used in the T. vaporariorum, this was evaluated for DVCV. Indeed, both T. abutilonea and T. vaporariorum transmit the virus with efficiencies of over 36 and 12%, respectively when plants were inoculated with 50 whiteflies after 48-h AAP and IAP . The disease was first identified in 1943 and has since been reported in Venezuela, Columbia, Ecuador, and Peru . The host range of the virus is rather restricted, and includes species in the genera Solanum, Polygonum, Rumex, Tagetes, Catharanthus, and Malva able to sustain virus replication whereas many common crinivirus indicators including Nicotiana, Datura, and Physalis species are resistant to infection . Sequencing of the genome of SPaV confirmed it as a crinivirus (Strawberries (family Rosaceae) are known to be natural hosts for about 30 viruses  , severainivirus . It continivirus .There have been reports of severe strains of the pallidosis agents that are lethal on indicators. Graft transmission of multiple isolates from the eastern and western United States caused only mild symptoms and it is most likely that these “severe strains” actually represented mixed virus infections involving not only a crinivirus, but likely another partner virus .Strawberry pallidosis associated virus is transmitted by T. vaporariorum, although somewhat inefficiently compared to BPYV . The decline epidemic was estimated to cause losses of about 50 million dollars for the two seasons ; incidence of SPaV was as high as 90% compared to 40% for BPYV. In plants from the Mid-Atlantic states that indexed positive for pallidosis disease based on symptoms, 37 of 38 plants were positive for SPaV and only about 25% were positive for BPYV  in Spain in 2003. Symptoms were similar to nutritional disorders with yellowing of the leaf blade, whereas pods appeared malformed. Leaves were brittle and whitefly transmission with B. tabaci Gennadius yielded reproducible symptoms. These observations pointed to a crinivirus infection. Confirmation came with the cloning of the HSP70h gene of the virus, which was named Bean yellow disorder virus  were able to sustain virus replication. Given the high incidence of the virus in greenhouses, control measures have primarily focused in these production systems. Beans grown in screenhouses had 14 times fewer whiteflies per plant. The incidence of the virus under screenhouse protection never exceeded 12.5% unlike that in conventional greenhouses which reached over 80% (Legumes (family Fabaceae) are infected by numerous viruses, several of which cause significant losses with many regularly identified in new locations around the world . This was BnYDV; . An extes BnYDV; . BnYDV gs BnYDV; . Phyloge. tabaci . Transmin a week . More thover 80% . Given tB. tabaci biotypes B and Q whereas limited sequence data revealed that the agent shared similarities with criniviruses (Cucurbit chlorotic yellows virus (CCYV), has a typical bipartite crinivirus genome, encoding four proteins in RNA1 and eight in RNA2  was initially discovered in the United Arab Emirates in 1982 . As with other criniviruses, symptoms are more prominent on older leaves with younger leaves remaining symptomless. CYSDV infections result in reduced plant vigor, and can significantly reduce fruit sugar production, resulting in poor tasting, unmarketable fruit. Islands . The virThe host range of CYSDV was originally believed to be restricted to members of the Cucurbitaceae ; howeverB. tabaci A, B, and Q . The virus is transmitted by B. tabaci biotypes A and B with similar efficiencies. Whiteflies can acquire and transmit the virus with AAP/IAP of 1 h each. Transmission was more efficient after 24 h of feeding whereas retention did not exceed 4 days. The host range includes at least 31 species belonging to 13 families, with several noteworthy hosts including spinach, sugar beet, and several weed species commonly found in the southwestern United States  was partially characterized at the biochemical level  was originally identified in 1996 from greenhouse-grown tomatoes (Lycopersicon esculentum Mill.) from Florida , although genetically the two viruses vary significantly. Several methods are now available to differentiate ToCV from TICV, including RT-PCR  and S. peruvianum L., as well as S. chilense  Reiche  Reiche . IntrogrLettuce infectious yellows virus is the most thoroughly studied virus in the genus Crinivirus. It was discovered in the southwestern desert agricultural regions of the United States in 1981 . Unlike most other criniviruses affecting commercial agriculture, which have effectively been distributed around the world, LIYV remained predominantly confined to southwestern United States and northern Mexico. This is due to its close relationship with the B. tabaci biotype A, which shared a common geographical range with the virus  with leaves becoming thickened and crispy, breaking easily when bent. Yield is affected through decreased fruit size and number  or crops associated with grafted transplants (CYSDV and CCYV). In today’s global trading environment there is constant germplasm exchange among individuals and organizations. The previous examples of crinivirus-driven epidemics should become lessons for the future and provide the impetus to improve plant certification schemes. This will facilitate increasing international trade in plant and plant products while decreasing the unintentional movement of plant pathogens. Given that some of the aforementioned viruses remain confined in specific geographic areas  it is still feasible to minimize their future impact by eliminating movement of infected material into areas where these viruses are not present. It is also important to establish vector exclusion strategies at the nursery or propagation field level. It has been common practice in certification schemes that plants are only visually inspected at the certified plant (G4) level. Using strawberry or blackberry as an example, neither BPYV, BYVaV nor SPaV cause symptoms in single infections in modern berry cultivars. However, when singly infected plants are planted in the field they often become infected with additional viruses and the resulting mixed infections can lead to serious epidemics. Exclusion and testing at the G4 level or prior to distribution can enhance longevity and profitability of the crops within regions and prevent or reduce accidental introduction of viruses into new production areas.Given the relatively recent identification of criniviruses as economically important disease agents, work has primarily focused on characterization, epidemiology, and in certain cases chemical control of vectors. Still, the ultimate control strategy for any pathogen is strong, stable genetic resistance. Resistance using modern methods such as RNA interference is probably the most straightforward and durable approach to prevent infection by viruses, but public resistance to genetically modified plants especially in crops that are labeled as “healthy food” or “superfoods” such as fruits and vegetables, the primary hosts for criniviruses, has minimized the application of this technology. For the majority of the criniviruses little or no work has been directed toward identification of resistance using traditional screening of germplasm resources and/or breeding to incorporate such sources into commercially acceptable cultivars. In the few cases where resistance has been identified it is almost always found in wild accessions, which requires many generations of backcrossing before the relevant genes are incorporated into marketable varieties. That is not to say progress is not being made. Sources of resistance to LIYV were identified in both lettuce and melon , 2000, aAmphorphora agathonica (Hottes) had been effective for over 50 years in controlling three aphid-borne viruses in raspberry in the North America, before new biotypes of the vector developed that overcame the resistance (Criniviruses are transmitted in a semi-persistent manner and chemical control of vectors has not always been effective for virus disease management. In addition, the development of resistance to insecticides in insect populations and the effect of insecticides on whitefly predators may have a negative impact on vector and virus control, particularly in systems using broad integrated pest management approaches. Consequently, it may be appropriate to consider a more generic approach, such as identification of resistance against whitefly vectors. There have been several cases in which insect resistance has been identified in plants . In manysistance . Forms osistance . Studiessistance , 2012. AThere have been numerous significant breakthroughs in understanding criniviruses, the diseases they cause, and their epidemiology. However, a great deal more work is needed on virus control, including an emphasis on certification to minimize virus movement, identification of resistance sources against both vectors and viruses, and introgression of resistance genes into commercially acceptable germplasm. These should be priority areas for long-term reliability of crinivirus management. Such efforts will complement or reduce the need for extensive pesticide-based programs, and will minimize the impact and spread of criniviruses in world agriculture.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cryptococcus gattii, which has affected hundreds of otherwise healthy humans and animals. Over the same time period, C. gattii was the cause of several additional case clusters at localities outside of the tropical and subtropical climate zones where the species normally occurs. In every case, the causative agent belongs to a previously rare genotype of C. gattii called AFLP6/VGII, but the origin of the outbreak clades remains enigmatic. Here we used phylogenetic and recombination analyses, based on AFLP and multiple MLST datasets, and coalescence gene genealogy to demonstrate that these outbreaks have arisen from a highly-recombining C. gattii population in the native rainforest of Northern Brazil. Thus the modern virulent C. gattii AFLP6/VGII outbreak lineages derived from mating events in South America and then dispersed to temperate regions where they cause serious infections in humans and animals.Over the past two decades, several fungal outbreaks have occurred, including the high-profile ‘Vancouver Island’ and ‘Pacific Northwest’ outbreaks, caused by  Geomyces destructansBatrachochytrium dendrobatidisCryptococcus gattii outbreaks in Canada  and the Pacific Northwest of the USA, as well as several case clusters of C. gattii infections in Western Australia, and Brazil During the past two decades, fungal outbreaks caused by previously rare genotypes or even novel species have emerged that affect humans, mammals and amphibians. Among them are the white-nose-syndrome in bats due to C. gattii, which expanded from Vancouver Island (Canada) to the mainland of British Columbia and the Pacific Northwest C. gattii was almost exclusively known as the cause of rare cryptococcal infections in otherwise healthy humans and animals living in tropical and subtropical regions, mainly in Australasia and South America C. gattii strains belonging to the AFLP6/VGII genotypic lineage C. gattii infections on Vancouver Island was estimated to be approximately 27 times higher than in Northern Australia where C. gattii is endemic C. gattii infection, which is a case-fatality rate of 8.7% C. gattii-related disease reported from captive psittacine birds in São Paulo, Brazil , caused by C. gattii complex C. gattii strains of the major genotype AFLP6A/VGIIa have a higher virulence potential than strains that belong to AFLP6B/VGIIb All these outbreaks and case clusters are caused by genotype AFLP6/VGII strains, one of the five known genotypes within the C. gattiiC. gattii propagules Trees are the natural habitat of C. gattii complex has been extensively studied and led to several hypotheses to explain the origin of the Vancouver Island outbreak C. gattii population demonstrated the highest genetic diversity and these authors suggested that the origin of this species may be in South America. Thus, the origin of the highly virulent lineages within C. gattii genotype AFLP6/VGII remains unclear.The molecular epidemiology of the C. gattii. Here we provide an evolutionary model for the origin of the C. gattii epidemics using extensive molecular population analyses that includes amplified fragment length polymorphisms (AFLP) analysis, a novel multi-locus microsatellite panel, and coalescence gene genealogy analysis based on data obtained from a novel multi-locus sequence typing scheme based on sequence characterized AFLP regions (SCAR-MLST). In addition, we performed additional sequencing and re-analyzed recently published MLST data C. gattii AFLP6/VGII strains represent the ancestral lineages within the global population, which is in agreement with the observed high genetic diversity at that locality. Coalescence gene genealogy analysis showed that the basal lineages within genotype AFLP6/VGII have a South American origin. Thus we provide strong evidence that South America is the cradle for the global population, including the expanding C. gattii AFLP6/VGII outbreaks in British Columbia (Canada) and in the Pacific Northwest of the USA Understanding the genetic and geographic origin of these outbreaks is critical in order to control the spread of C. gattii AFLP6/VGII was assessed by application of coalescence gene genealogy analysis using the SCAR-MLST dataset which was analyzed after removing 59 homoplasious sites in the 3,357 nucleotides alignment which resulted in 32 haplotypes  (6 gbp. The estimated divergence time of the two closely related C. gattii strains A1M-R265 and CBS7750 (both lineage H01) was found to be approximately 51,449 years based on the number of synonymous nucleotide substitutions observed across 4,771 protein-coding orthologous genes.Generation time scaled coalescence analysis revealed that the major clade  and the basal clade (H17 and H24) emerged approximately 17×10nt (gbp) ; Table 1C. gattii case cluster among psittacine birds in Brazil (H02) emerged around 4.2×106 gbp. A major diversification event that gave rise to a global lineage (H03) occurred approximately 5.3×106 gbp and includes strains from the C. gattii outbreak in the Pacific Northwest and strains belonging to the minor genotype from the Vancouver Island outbreak. The latter were found to be genetically indistinguishable from strains obtained from a case cluster among over 100 sheep in Western Australia based on the consensus multi-locus sequence typing scheme for C. neoformans and C. gattiiThe lineage representing the 6 gbp a lineage dispersed to Africa, followed by a massive dispersal and recombination event approximately 5.3×106 gbp that introduced C. gattii AFLP6/VGII into Australasia, Europe and North America , followed by AFLP (D = 0.928) and SCAR-MLST (D = 0.909). Using the individual AFLP, SCAR-MLST and STR datasets the South American population had the highest genetic diversity, which was corroborated when these datasets were combined resulting in 58 genotypes among 76 strains (D = 0.951) (G0) for each of the datasets, resulting in an overall significantly higher G0 of 36.10 (P<0.05) compared to the other four populations, supporting a South American origin of the global C. gattii AFLP6/VGII population  . The Soupulation .C. gattii genotypes in South America C. gattii AFLP6/VGII population has a higher influx than efflux of migrants from the other populations with South America being the main contributor, followed by Europe, Australasia and Africa  emerged from a diverse cluster containing Australasian , European (ST17) and South American  strains  when compared to other strains (P<0.001) less virulent than strains with the major genotype AFLP6A/VGIIa from the Vancouver Island outbreak region, which had ST50 values ranging from 3.9±0.1 to 6.8±0.3 days  in the South American population suggests that recombination events are likely to occur at this locality. The majority of these mating-type a strains sampled from various localities in Colombia were observed to be clonal. STE12 mating-type analysis of the C. gattii AFLP6/VGII strains confirmed that the population in Mediterranean Europe contained mating-type a and α strains as well, while the African, Australasian and North American populations were mating-type α only  in the current population of C. gattii AFLP6/VGII corroborated the ancestral recombination graph analysis results, indicating a high number of recombination events (RM = 22) in South America versus those in the other populations   but not within the extended ‘Fraser and Byrnes’ MLST dataset (P = 0.196). When the SCAR-MLST data was split into the five populations, evidence for recombination was found by the ΦW test in the Australian (P = 0.033), European (P = 0.044), North American (P<0.001) and South American (P<0.001) populations. CASS network analysis also revealed highly supported signs of actual recombination in the SCAR-MLST dataset  . The ΦW  dataset . Thus, c dataset .C. gattii AFLP6/VGII population has most likely a South American origin rather than being introduced from another geographical locality such as Australia, Africa or Europe, as was proposed in previous studies C. gattii AFLP6/VGII in the natural and pristine environment of the Amazon rainforest Eucalyptus seeds containing C. gattii, as suggested Our study demonstrates that the current global C. gattii AFLP6/VGII populations pairs showed that South America is the major donor of migrants for the other populations, with Africa being the exception due to a very small influx of genetic material into South America, while this populations seems not to contribute to the genetic influx into the African population , as opposite to the other populations were mating-type a strains were absent or outnumbered by mating-type α strains, an indication that regular mating events play a more important role in the South American population than so-called same-sex mating C. gattii AFLP6/VGII case cluster among psittacine birds in Brazil is genetically not closely related to any of the other outbreaks , the Pacific Northwest (USA), case clusters among psittacine birds in Southern Brazil, an epidemic among sheep in Western Australia, and a higher prevalence of infections among immunocompetent native children in Pará state, North East Brazil C. gattii (AFLP4/VGI) emerged in the European Mediterranean area where it caused several small outbreaks among goats C. gattii AFLP6/VGII infections were observed in Greece, and a recent survey revealed that C. gattii AFLP4/VGI occurs widespread in indigenous tree species in the Spanish Mediterranean region C. gattii is moving from its endemic tropical and subtropical regions towards more temperate climate zones, such as the European Mediterranean region During the past two decades several C. gattii strains with the major genotype AFLP6A/VGIIa are highly virulent in two host models, irrespective of their clinical, veterinary or environmental source  and the Pacific Northwest (genotype AFLP6C/VGIIc), but is present among all globally sampled coalescence gene genealogy lineages, including the most basal lineages from South America. Non-virulent strains co-occurred with virulent ones in the Vancouver Island outbreak lineage H01  and in full compliance with the Israel animal welfare act (law 5754–1994 and 5761–2001). The study was approved by the joint ethics committee for animal welfare (IACUC) of the Hebrew University and the Hadassah Medical Centre . The facilities of the Hebrew University are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (accreditation number 1285), experiments were performed in full compliance with national and international guidelines.C. gattii strains was selected based on previous PCR fingerprinting and AFLP genotype analyses and included strains involved in the Vancouver Island outbreak , the Pacific Northwest outbreak AFLP6C/VGIIc, the case cluster among psittacine birds in Brazil, strains from Western Australian, and strains obtained from pediatric cryptococcosis cases , Australasia (n = 38), Europe (n = 6), North America (n = 48) and South America (n = 76) (d-glucose and 2% w/v agar) at 25°C and stored at 4°C on slants or in glycerol stocks at −196°C in liquid nitrogen.A global set of 178 (n = 76) . Eleven C. gattii AFLP6/VGII strains, indicated in 2O to lyse macrophage cells. After 30 minutes, the intracellular yeast were released and collected and another 200 µl dH2O was added to collect the remaining yeast cells. Intracellular yeast were then mixed with Trypan Blue at a 1∶1 ratio and the live yeast cells were counted. The IPR value was calculated by dividing the maximum intracellular yeast number (either at 18 or 24 hours) by the initial intracellular yeast number at T = 0. Each assay was repeated at least three times on different occasions.A subset of C. gattii strains was studied in a BALB/c mice model as described by Ma et al. 6 yeast cells from a 48 h culture on Sabouraud Dextrose agar at 30°C) in PBS were injected into the tail veins of male albino BALB/c mice (body weight of 20–23 g). The tail vein injection method was preferred above the use of the inhalation model since the former method provides a more controlled measurement of the number of yeast cells entering the blood system compared to the inhalation model. A hemocytometer was used to determine the concentration of yeast cells, while the viable count was measured as the number of CFU on Sabouraud Dextrose agar plates after 24–48 h of incubation at 30°C. For each of the tested C. gattii strains, a set of 8–10 mice were included, based on IPR outcome of the inoculated yeast strain, that were maintained in separate cages. The genome sequenced reference strain A1M-R265 was used as a control for each experiment. The survival rate of the mice was recorded daily, for up to 45 days, in such a way that the animal handlers were unaware of the corresponding IPR values for the strains tested. Statistical analysis was performed using the Kaplan-Meier method in SPSS v21 .Virulence of the myo-inositol, typically assimilated by tremellaceous yeasts, was chosen for the generation experiment since this is an important factor for the sexual development and virulence potential of Cryptococcus, and it is abundantly present in the human central nervous system Strains were cultivated for 48 h on malt-extract agar at 25°C. The carbon source C. gattii strains  and CBS6956 (αB) as a reference. Strains were genotyped using various approaches as described in Genomic DNA was extracted as described in SCAR-MLST and ‘Fraser & Byrnes’ MLST datasets were subjected to various sequence based analyses that include a search for the best fitting nucleotide substitution model, bootstrapped Maximum Likelihood phylogenetic analysis, genetic diversity analyses, recombination analyses, ancestral recombination graph analysis and coalescence gene genealogy analysis. The detailed workflow for these analyses is described in C. gattii AFLP6/VGII strains the environmental and non-virulent C. gattii strain CBS7750 and the virulent clinical reference strain A1M-R265 were subjected to whole genome sequencing with an approximately 350 fold coverage of the genomes. The workflow that consists of the MIRA mapping assembly, de novo assembly using SOAP, SNP and indel detection using Varscan and the genome-wide divergence time estimate is provided in To estimate the divergence time of two closely related Figure S1Microsatellite analysis of C. gattii AFLP6/VGII strains. Minimum spanning tree analysis based on ten microsatellite loci showing the genetic diversity of the complete set of C. gattii AFLP6/VGII strains and especially that of the South American population (red). Case cluster and outbreak related clusters are indicated. Colours represents the populations of Africa (yellow), Australasia (blue), Europe (purple), North America (green) and South America (red). Circle sizes are relative to the number of strains that are represented by that microsatellite type. Connecting lines between circles indicate the number of different microsatellite between both groups. Thick black lines represent one different locus, thin black two loci, dashed black line three loci, dashed dark grey line four loci and five or more different loci are indicated with dashed light gray lines plus number of different loci.(TIF)Click here for additional data file.Figure S2Neighbour Joining analysis of arbitrarily scored AFLP markers. The matrix of absent (0) and present (1) differential AFLP markers , AUS , EUR (Europe), NA (North America) and SA (South America). markers  has been(TIF)Click here for additional data file.Figure S3Phylogenetic analysis of the extended ‘Fraser & Byrnes’ MLST dataset. Unrooted Maximum Likelihood phylogenetic analysis of the sequence types found within the concatenated dataset of seven nuclear MLST loci as provided by Byrnes et al. [S14] and Fraser et al. [S4] and extended with additional strains in the current study. The most ancestral lineage (here ST07) from the SCAR-MLST based coalescence gene genealogy analysis is indicated in this figure with a tree to highlight its origin from pristine Amazon rainforest. When rooted with C. gattii genotype AFLP4/VGI, AFLP5/VGIII and AFLP7/VGIV, strains within ST18 are basal to the other lineages. The outbreaks in North America are indicated with icons of human, cat and dolphin, and the parrot in the South American population highlight the case cluster of C. gattii infections among psittacine birds in Southern Brazil. Populations are indicated behind strain and haplotype numbers as AFR (Africa), AUS , EUR (Europe), NA (North America) and SA (South America).(TIF)Click here for additional data file.Figure S4Ancestral recombination graph analysis based on SCAR-MLST. The ancestral recombination graph shows historical recombination events that gave rise to the current population structure represented by the clone corrected 49 sequence types (STs) observed within the concatenated nuclear SCAR-MLST dataset (C. gattii AFLP6C/VGIIc Pacific Northwest outbreak (ST39) has been indicated with a human and cat symbol. The tree icon indicates the most ancestral lineage that was isolated from a tree in the pristine Amazon rainforest (ST26). The 167 blue circles indicate historical recombination breakpoint positions within the concatenated nuclear SCAR-MLST dataset. dataset . The ST0(TIF)Click here for additional data file.Figure S5CASS network analysis based on SCAR-MLST. Recombination network analysis based on the CASS algorithm shows that several recombination events occurred in the current global population and that the majority of parental donors came from the South American population. Populations are indicated behind strain and haplotype numbers as AFR (Africa), AUS , EUR (Europe), NA (North America) and SA (South America). The outbreaks in North America are indicated with icons of human, cat and dolphin, and the parrot-icon in the South American population highlight the case cluster of C. gattii infections among psittacine birds in Southern Brazil.(TIF)Click here for additional data file.Figure S6Growth curves of C. gattii AFLP6/VGII strains on myo-inositol. Growth curves of the three replicated growth rate experiments for A1M-R265 (major genotype AFLP6A/VGIIa Vancouver Island outbreak lineage) and CBS1930 (Aruba). The formula for each of the trendlines is provided as this is part of the formula to calculate the generation time. For the coalescence gene genealogy generation time scale calculation, the average value is used from all six growth rate experiments.(TIF)Click here for additional data file.Table S1Map of informative sites used for the coalescence gene genealogy analysis. The clone corrected SCAR-MLST data has been collapsed into haplotypes after removal of homoplasious sites. Informative sites within the complete dataset, and among the 32 haplotypes identified, are provided per nuclear SCAR-MLST locus that has been provided as the Fragment-number. IGS1 refers to the Intergenic Spacer 1 region (see gion see . Colours(PDF)Click here for additional data file.Table S2Population sizes and historical migration rate estimates among Cryptococcus gattii populations. The population sizes and migration rates were estimated using Migrate v2.3 (http://popgen.scs.fsu.edu) based on the five nuclear SCAR-MLST loci. The centre column shows the mean Nmµ value, while the left flanking values represents the lower  and upper  95% confidence interval values.(PDF)Click here for additional data file.Table S3List of strains and background information.(XLSX)Click here for additional data file.Table S4Overview of molecular variation, including number of haplotypes, nucleotide diversity, estimates of theta based on the number of segregating sites and recombination parameters. The genetic diversity is provided per population and for all strains, as well as for each locus independently and the mean value for the given values. Given values are for the number of sites within an alignment with or without alignment gaps, the nucleotide diversity per site (π) corresponding to the average number of nucleotide differences per site between two sequences, the number of segregating or polymorphic sites (S), the Watterson’s estimated θ per locus and per nucleotide, the ‘Hud4Nc per site’ value representing the recombination rate per generation between the most distant nucleotides. The last two values listed the number of recombination events based on the ‘four gametic test’ and the minimum number of recombination events in the history of the sample (Note that provides an underestimation of the number of recombination events).(PDF)Click here for additional data file.Table S5Primers used for SCAR-MLST and microsatellite typing.(PDF)Click here for additional data file.Materials and Methods S1Supporting Information.(DOCX)Click here for additional data file.


Tobacco mosaic virus (TMV) requires three processes that repeat over time: initial establishment and accumulation in invaded cells, intercellular movement, and systemic transport. Accumulation and intercellular movement of TMV necessarily involves intracellular transport by complexes containing virus and host proteins and virus RNA during a dynamic process that can be visualized. Multiple membranes appear to assist TMV accumulation, while membranes, microfilaments and microtubules appear to assist TMV movement. Here we review cell biological studies that describe TMV-membrane, -cytoskeleton, and -other host protein interactions which influence virus accumulation and movement in leaves and callus tissue. The importance of understanding the developmental phase of the infection in relationship to the observed virus-membrane or -host protein interaction is emphasized. Utilizing the latest observations of TMV-membrane and -host protein interactions within our evolving understanding of the infection ontogeny, a model for TMV accumulation and intracellular spread in a cell biological context is provided.Successful systemic infection of a plant by  Tobacco mosaic virus (TMV) encodes four known functional proteins: the 126 and 183 kDa replication-associated proteins, the movement protein (MP), and the structural capsid or coat protein (CP). In order to have a successful infection, these four multifunctional proteins cooperate with many host components. The host membrane and cytoskeleton are sub-cellular structures important for TMV infection. TMV-induced granules or inclusion bodies that contain membranes also contain host proteins. In this review, we discuss the changing roles of host membranes, cytoskeleton, and inclusion body-associated proteins as infection progresses. Findings reported in the literature are first presented in the section(s) where the effect on virus physiology was observed rather than where it may additionally influence this activity. For example, the influence of synaptotagmin on TMV physiology . TMV begins to disassemble within 3 min after entry and disassembly of CP from the capsid is associated with translation of viral RNA  did not prevent granule formation, although they were smaller and less stable. The granules were shown to associate with fluorescently labeled ER. The 5′ methylguanosine cap on the vRNA was necessary to anchor vRNA to the ER/actin complex: absence of the cap leading to vRNA degradation and no granules  use their parental genomes to synthesize complementary negative strands which serve as templates for the synthesis of progeny full-length positive strands and subgenomic mRNAs containing MP and CP open reading frames . The ER was implicated as a site for virus replication complex (VRC) formation through co-localization of an ER marker, BiP, with MP-GFP and in turn, MP-GFP co-localization with replication proteins during immunofluorescence studies .es ORFs; . Althouges ORFs; . The 126 studies . Reichel studies . Interes studies . This fiTobacco mild green mosaic virus (TMGMV), form different subcellular structures containing replication proteins late in the infection cycle: TMV forming X-bodies and ToMV and TMGMV forming virus bundles when analyzed through immunocytochemical EM studies (Turnip vein clearing virus (TVCV), X-bodies are rare .The host proteins within VRCs or inclusions that contain them are not fully identified. Late in infection, TMV-induced X-bodies have been shown to contain the microtubule component, β-tubulin, through immunocytochemical EM studies . The function of this protein in X-bodies is unknown, but perhaps it could be to aid the degradation of body components analogous to the suggested function of microtubules during TMV MP turnover . The hosTobacco mosaic virus-Cg (crucifer-infecting virus), in a yeast two-hybrid screen . This interaction is likely important for VRC formation, but the co-localization of TOM1 and tobamovirus replication proteins in live cells has not been reported.Tobamovirus multiplication 1 (TOM1) is a predicted multi-pass transmembrane protein required for tobamovirus accumulation . Surprisd screen . This ind screen . The repd screen . It is hFor TMV to establish a systemic infection, the virus or its components must move within a cell to establish an infection site, multiply and finally position for movement to the next cell. The granules of vRNA that form on initial infection, the VRCs that form during infection and the 126 kDa protein- and MP/vRNA-containing inclusions observed during ectopic expression all move within the cell e.g., . These cFigure 1). Indeed, vRNA has been visualized in the perinuclear bodies by bimolecular fluorescence complementation using a modified sequence-specific RNA-binding protein, Pumilio1, or by classical in situ RNA labeling , although evidence against a direct action for microfilaments exists and microtubules may not be in an intact form or the required microtubule array is unusual in that it is impervious to certain pharmacological agents . Callose is present in the neck region of the PD . Cucumber mosaic virus altered in its ability to target the PD. This mutant MP, which also has severing activity, fragments microfilaments in the cytoplasm . Interactions of the TMV MP with other host proteins that influence TMV spread have been described . These findings support those using a mutant strain of TMV whose temperature sensitive intercellular movement is correlated with the temperature sensitive localization of MP with microtubules .Regarding microtubules and TMV intercellular movement, there is evidence that MP interaction with the microtubule end-binding protein 1a (EB1a) is important for TMV spread. otubules . Howeverotubules . Additiootubules . These aotubules , led to otubules . These aMicrofilaments have been shown to be important for TMV intercellular movement, but the interpretation of their involvement in this activity is evolving. Findings from early studies using a GFP-labeled virus showed that disruption of microfilaments, through pharmacological methods or by silencing actin, inhibited sustained (2 days and beyond post-inoculation) virus intercellular movement . This inFigure 1). On treatment with LatB or silencing of myosin XI-2, the TMV VRCs would remain at the cell wall/PD interface, inhibiting normal cell–cell communications. This stress would be signaled to the cells in advance of the virus spread and modify these cells to inhibit subsequent virus spread. This hypothesis is supported by the finding that the VRCs of TMV move away from the cell wall as the infection passes . This hypothesis, suggesting a requirement for actomyosin-mediated vesicle trafficking of the VRC or VRC components from the wall membranes for sustained virus movement, can be evaluated through cell biological studies.Irrespective of the mechanism of early movement between cells by TMV, an explanation of this virus’s requirement for microfilaments for sustained movement is required. n passes . This inn passes  and the n passes . The train vitro . Synaptoembranes . A dominembranes . The dirNicotiana tabacum and its expression levels were positively correlated with MP accumulation, VRC numbers and virus spread .Regarding non-cytoskeletal or membrane-associated proteins and TMV intercellular movement, a gene encoding a class II KNOTTED1-like protein, NTH201, was cloned from s spread . NTH201 s spread . SilenciN. benthamiana genome (Cell biological studies over the last 20 years have tremendously aided our understanding of TMV accumulation and spread. Without advanced molecular and biochemical technologies allowing virus and virus component labeling and advanced imaging hardware our understanding of the individual processes during virus spread would be diminished. For example, if virus intercellular movement were studied by genetics alone the importance of the transport of TMV vRNA granules to the perinuclear region of the ER versus the transport of TMV VRCs and MPs to the PD could go unrecognized. This said, some cell biological studies have yielded conflicting or controversial results. This is especially true of pharmacological studies and researchers must carefully control as many variables in these studies as possible. In addition, conclusions from pharmacological studies should be verified using other methods. Use of novel virus labeling techniques and advanced microscopes will allow further advances in this area. For example, the identification of small fluorescent tags that do not influence the function of the viral protein to which it is fused will be helpful. The iLOV protein, derived from the blue light receptor phototropin and much smaller than GFP, has been available for some time and was a first step toward utilizing smaller fluorescent tags . More rea genome  will allThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.