Diagnostic of begomoviruses in complex infections –a case study

Complexes of viruses inducing a syndrome in plants strongly hinder the identification of the causal agent of a disease. The Sida micrantha mosaic disease is associated with a complex of begomoviruses. For more than twenty years, two DNAs A (DNA A2 and DNA A3) belonging to this complex could neither be detected nor isolated from Sida micrantha Schr. plants, although one of them (DNA A2) now appears to be the major component of the complex. A random unintended Bemisia tabaci-mediated transmission of begomoviruses from several Sida species – including S. micrantha – to experimental Malva parviflora plants resulted in the serendipitous finding of these new DNAs A. Simultaneously, a number of other begomoviruses infecting Sida plants from several Latin American countries were transmitted to M. parviflora plants and the convergence of them resulted in natural pseudorecombinants. Pseudorecombination, however, took place exclusively between heterologous genomic components that shared identical binding sites for the replication-associated protein AC1. This case study constitutes an exceptional opportunity for the analysis of plant–virus– vector interactions in a quasi-natural environment. In addition to that, the methodology described here may be used to isolate and characterize different begomoviruses inducing a syndrome during mix infections.


Introduction
In several aspects, geminiviruses (Rybicki et al., 2000) are intriguing pathogens of plants. From an economical perspective they are responsible for extensive losses in diverse cash crops.
For instance, African cassava mosaic (Thottappilly, 1992), tomato leaf curl (Czosnek and Laterrot, 1997), cotton leaf curl (Briddon and Markham, 2001), and bean golden mosaic (Morales and Anderson, 2001) diseases have been major bottlenecks in the production of vegetables and fibber crops worldwide during the last three decades.
Because of their limited coding capacity, geminiviruses strongly rely on host replication complexes to accomplish their life cycle. This peculiarity makes them interesting models for the study of replication, transcription and cell cycle regulation in higher plants (Hanley-Bowdoin et al., 1999). Replication of geminiviruses is assumed to occur through a rolling circle mechanism (RCR) (Hanley-Bowdoin et al., 1999) complemented by a recombination-dependent (RDR) mechanism (Jeske et al., 2001).
The and a cell-to-cell movement proteins (BC1) (Rybicki et al., 2000). Since DNA B does not possess a replication-associated protein, its replication depends on the DNA A-encoded AC1 protein and takes place in a virus-specific manner. While all determinants of such specificity are not known at the moment, it has been demonstrated that a direct repeat located in the CR and matching the consensus sequence 5'-GG-AGTAYYGG-AG is necessary, although not sufficient, to confer specific AC1 binding to DNA B (Hanley-Bowdoin et al., 1999). Thus, the existence of a DNA B component implies the presence of its cognate DNA A.
Sida micrantha mosaic disease is associated with a complex of begomoviruses that have been partially characterized (Jovel et al., 2004;Jovel et al., 2007). Unraveling the first pieces of this puzzle has been a 25-years-long effort. Initially, a DNA A (DNA A1) and a DNA B (DNA B1) were cloned and sequenced. The weak homology between DNA A1 and DNA B1 in the common region

Plants and viruses
S. micrantha plants harbouring the Sida micrantha mosaic viruses have been described (Abouzid and Jeske, 1986) and will be referred to in this paper as "original S. micrantha plants".

Extraction of Nucleic Acids
One young and symptomatic leaf was shock frozen in liquid nitrogen, ground to powder, and shaken for 15 min at room temperature (RT) with 500 µl homogenization buffer (100 mM Tris-HCl, pH 7.5; 1 mM EDTA; 100 mM NaCl; 0.6% SDS; 100 mM DTT) plus an equal volume of phenol-chloroform (PC, 9:1). The suspension was centrifuged for 5 min at RT (14000 rpm). The supernatant was extracted with a volume of chloroform for 5 min, centrifuged 5 min as above, and finally precipitated with one tenth of 3M NaAc (pH 4.8) and two volumes of ethanol (EtOH), overnight at -20ºC. After centrifugation (30 min, 14000 rpm, 4ºC), the pellet was washed with 0.5 ml 70% EtOH, recovered by centrifugation (5 min, 14000 rpm, RT), dried at RT and re-suspended in 50 µl of 1x TE buffer (10 mM Tris-HCl, 1 mM Na-EDTA, pH 7.5).

Labelling of probes and hybridization
The AC1/AC3 (primers SmRep-V and SmRep-C;  2D). This plant contained only low concentration of viral DNA ( Fig. 2A), exhibited mild symptoms, and recovered after six weeks.
A peculiar combination was found in plant 551. As mentioned above, the DNA B of SiGMCRV was clearly identified in this plant ( Fig. 2B). However, after the Alu I-RFLP analysis (Fig. 2E and DNA B1 (Fig. 7B), suggesting that they are cognate components of a third begomovirus in the SimMV complex. We have called this new molecule DNA A3.

Mixed infections originating a syndrome
Cloning and characterization of the SimMV has been a 25-years-  (Eigen, 1993), however, this circumstance should be considered to define the causative agents of syndromes. Perhaps it is therefore necessary in future to combine many independent molecular clones of all DNA components to restore the biological variability and to reproduce the full syndrome in Sida plants.  (Fig. 6).
Moreover, previous studies have established that the different biotypes of B. tabaci show no preferences for the transmission of begomoviruses (Bedford et al., 1994).  et al., 1998). Therefore, a conclusive resolution of the causal agent of the Sida micrantha mosaic will await until the Koch's postulates could be fulfilled.

Natural pseudo-recombinants
Several independent lines of evidence suggest that the AC1 protein of begomoviruses is a DNA-binding protein with nicking and closing activities and acts in a virus-specific manner (Hou and Gilbertson, 1996;Orozco et al., 1998). The specificity is partially determined by a direct repeat preceding the TATA box for complementary transcription, which is identical between cognate components of a begomovirus but divergent in heterologous ones. The repeats match the sequence 5'-GG- (Hanley-Bowdoin et al., 1999). In the case of DNA A2 and DNA B2 of SimMV the common binding motif is GGGGtaatGGGG.

AGTAYYGG-AG
Pseudorecombination is a type of reassortment which occurs between two different begomoviruses that exchange their viral chromosomes (Unseld et al., 2000b).
In this paper, we showed that DNA A1 and DNA A2 of SimMV were able to transreplicate only