Rhizophagus irregularis.Phosphorus (P) is one of the most important nutrient elements for plant growth and development. Under P starvation, arbuscular mycorrhizal (AM) fungi can promote phosphate (Pi) uptake and homeostasis within host plants. However, the underlying mechanisms by which AM fungal symbiont regulates the AM symbiotic Pi acquisition from soil under P starvation are largely unknown. Here, we identify a HLH domain containing transcription factor RiPho4 from To investigate the biological functions of the RiPho4, we combined the subcellular localization and Yeast One-Hybrid (Y1H) experiments in yeasts with gene expression and virus-induced gene silencing approach during AM symbiosis.RiPho4 encodes a conserved transcription factor among different fungi and is induced during the in planta phase. The transcription of RiPho4 is significantly up-regulated by P starvation. The subcellular localization analysis revealed that RiPho4 is located in the nuclei of yeast cells during P starvation. Moreover, knock-down of RiPho4 inhibits the arbuscule development and mycorrhizal Pi uptake under low Pi conditions. Importantly, RiPho4 can positively regulate the downstream components of the phosphate (PHO) pathway in R. irregularis.The approach during AM symbiosis. The results indicated that In summary, these new findings reveal that RiPho4 acts as a transcriptional activator in AM fungus to maintain arbuscule development and regulate Pi uptake and homeostasis in the AM symbiosis during Pi starvation. AMF havterfaces . In retuterfaces . These bterfaces .Soil available P can be acquired at the root periphery and utilized by plants in the form of inorganic orthophosphate (Pi), however, Pi is always insufficient in the fields due to its low solubility and relative immobilization in soils . The forPHOSPHATE TRANSPORTER 1) family that are expressed in the ERM and IRM have been identified and characterized from some AMF species, for example, GmosPT, GigmPT, GvPT, and RiPT from Glomus mosseae, Gigaspora margarita, Glomus versiforme (currently Diversispora epigaea), and Rhizophagus irregularis  domains participate in the Pi export process at the symbiotic interface, releasing Pi into the PAS  signaling pathway between AMF and yeasts  . After t the PAS . Pi in t the PAS . On the  the PAS . The Pi d Pho91p . This sud yeasts .R. irregularis and GintSTE from Glomus intraradices (currently R. irregularis) are preliminarily investigated  play crucial roles in the regulation of gene expression in fungal cells and determine the functions of eukaryotic cells . Recent stigated , while tegularis . It is weostasis . Althougeostasis , 2022, tEucalyptus species is the most valuable and widely planted hardwood in the world , which contains a C-terminal bHLH domain, and provide experimental evidence for roles in the regulation of Pi uptake and homeostasis during AMF-Eucalyptus symbiosis. Moreover, our findings offer new insights into the control of Pi uptake and metabolism in the AM fungal symbionts at the symbiotic interfaces.After the investigation of Pi uptake and transport processes during AMF and plant interaction, to further understand the regulatory mechanisms of the Pi uptake and homeostasis at the symbiotic interface, we start to search the regulators (TFs) in R. irregularis DAOM 197198, which was propagated in the pot cultures with maize (Zea mays). Spores of R. irregularis were collected from Z. mays root segments. The plant material used in this study was Eucalyptus grandis . The surface-sterilization of E. grandis seeds was performed as the described previously (R. irregularis (about 200 spores per plant). After inoculation, E. grandis plants are cultivated in a growth chamber under 16 h: 8 h, 24°C: 19°C, light: dark conditions .AM fungus used in this study was eviously . The seeEucalyptus grandis plants were cultivated in pots under AMF inoculation (AM) and uninoculated control (NM) treatments. Each treatment was carried out with three Pi concentrations including 30, 300 and 1,000 μM K2HPO4   for 24 h. And the yeast cells were cultured in YNB/-Ura medium for 12 h, supplemented with different Pi concentrations  .R. irregularis, the genes of PHO signaling pathway in S. cerevisiae was used as the queries to search for the homologues genes in the genome of R. irregularis DAOM 197198  and symbiotic tissues  of R. irregularis download from DDBJ database. The accession numbers of RNA-seq reads are as follows: germinating spores harvested at a week after inoculation (DRA002591), germinating spores collected at 7 days after induction (GSE67913), laser microdissected cells contain IRM and arbuscules collected from Medicago truncatula mycorrhizal roots (GSE99655), mycorrhizal roots of M. truncatula , ERM collected from carrot root culture (GSE99655)  , 2020.R. irregularis was referred to the method of RiEF1α gene from R. irregularis was used as an internal control for qRT-PCR analysis. Relative expression levels were calculated using 2−ΔΔCt method. The list of gene-specific primers used for qRT-PCR analysis is given in The extraction of the genomic DNA from RiPho4 was amplified by gene-specific primers containing the BamHI site . Then the constructed vectors were transformed into the Y1HGold yeast strain, and grown on YNB/-Ura solid medium with Aureobasidin A (AbA) concentration (100 mM) for testing the minimal inhibitory concentration of AbA for bait-specific pAbAi plasmids. We transformed the bait-specific pAbAi fragments and pGADT7-RiPho4 were co-transformed into yeast cells, and screened using the SD medium lacking Leucine. The yeast cells (OD600 = 0.2) containing both RiPho4-pGADT7 and promoter fragments were inoculated on SD medium lacking Leucine with different AbA concentrations (100–800 ng/ml). The yeast cells carrying pAbAi-p53 and pGADT7-SV40 were used as the positive control, whereas the yeast cells containing pGADT7-RiPho4 and promoter fragments lacking the cis-element served as the negative control. In addition, the inhibitory effect was adjusted based on the growth of yeast (The ORF of e pGADT7 . To conset genes  (listed et genes  were synof yeast .Nicotiana benthamiana) is used for this virus-induced gene silencing (VIGS) experiment (RiPho4 from the-9 to +226 regions (RiPho4-RNAi-1) and + 1,200 to +1,429 regions (RiPho4-RNAi-2) relative to the start codon ATG (pTRV2-RiPho4-1 and pTRV2-RiPho4-2 were separately transformed into Agrobacterium tumefaciens GV3101 (A. tumefaciens culture (OD600 = 1.0) with pTRV1 and that with pTRV2 or pTRV2-RiPho4 were mixed together and activated by adding 10 mM Acetosyringone, then stood in darkness for 2 h. The inoculums were injected into the leaves of N. benthamiana, whose roots had been inoculated with R. irregularis for 4 weeks as described by Tobacco  at 37°C for 30 min . The colonization levels of SDH, ACP and ALP enzyme activity staining was calculated under the light microscope .E. grandis were lyophilized for 6 h. To grind dried samples into powder, we added magnetic beads to them and grinded them with a grinder (35 Hz) for 2 min. 0.3 g of sample powders were digested by 6 M nitric acid under the water bath at 90°C for 1 h . Total P concentrations of N. benthamiana were detected by the tissue total P content detection Kit  and measured with the Microplate Reader .Fresh samples of  for 1 h . The digcis-acting element by searching for 1.5 kb promoter sequences of the target gene coding regions through the NCBI database and PlantCARE software.The BLASTPThe unrooted phylogenetic tree of RiPho4 protein and other amino acid sequences in different fungi species were constructed with MEGA7.0  using thp < 0.05 was considered to be statistically significant. The different letters indicate significant differences among phenotypes or treatments. Data are preliminarily accounted by Microsoft EXCEL 2016, and statistical significances between treatments were analyzed by analysis of variance (ANOVA) using SPSS software . The Duncan’s multiple range test were used for comparing more than two datasets. A value of Sequence data from this article can be found in the AM fungal genome and GenBank libraries under the following accession numbers: RiPho4 (XP_025175129.1), RdPho4 (RGB28534.1), RcPho4 (GBB98521.1), GcPho4 (RIA86088.1), GmPho4 (KAF0357978.1), GrPho4 (RIB17793.1), DePho4 (RHZ83467.1). Other accession numbers of fungal Pho4 proteins were provided in E. grandis subjected to different Pi conditions, we treated mycorrhizal (AM) E. grandis with three phosphate concentrations when compared with non-mycorrhizal (NM) E. grandis. After 7 weeks inoculation, the growth performance of AM E. grandis was better than NM plants under medium and low phosphate conditions (30 and 300 μM) (E. grandis exposed to low Pi (30 μM) were significantly higher than those grown under the medium and high Pi treatments  (R. irregularis can promote the E. grandis Pi uptake and utilization efficiency to improve the plant growth under Pi-deficient conditions.To study the effects of AMF inoculation on the growth and Pi uptake of  300 μM) . Overall 300 μM) –D. Compa 300 μM) . In addi,000 μM) . Moreove,000 μM) . Activit,000 μM) . Taken tR. irregularis in mycorrhizal E. grandis roots during different Pi conditions. As shown in RiPho2, which was predicted to be a cofactor for RiPho4 , whereas RiVTC2 expression was not significantly enhanced in roots exposed to low Pi concentration , while the region from +436 to +515 is a HLH (helix loop helix) domain  domain , which i) domain . Further) domain  showed tR. irregularis and other different fungi species, we performed the phylogenetic analysis and conserved motif identification. The result shows that RiPho4 is related to AM fungal TFs GmPho4, GcPho4, FgPho4 and FmPho4, and has closely relative to the RdPho4 and RcPho4 from Rhizophagus diaphanous and Rhizophagus clarus . The qRT-PCR experiment was then performed on the mycorrhizal roots of E. grandis subjected to different Pi concentrations as mentioned above. As a result, the expression levels of RiPho4 were significantly inhibited in roots during high Pi concentrations   . This re,000 μM) , suggestS. cerevisiae imports into the nucleus under low phosphorus supply, whereas the Pho4 exports into the cytoplasm nucleus of yeast cells exposed to high phosphorus conditions , while this fusion protein was found in the cytoplasm of yeast cells exposed to high P concentration (10 mM) (Previous studies have found that ScPho4 protein in nditions . To inve (10 mM) . To furt (10 mM) . In concRiPho4 in AM symbiosis, we knocked down RiPho4 expression by the VIGS method. We designed two RNAi regions to target the corresponding specific regions of RiPho4 that was very divergent from the other genes containing bHLH domains in R. irregularis to avoid off-target silencing . The results showed that the transcriptional levels of RiPho4 in the VIGS-RiPho4 roots were significantly reduced by more than 60%, when compared with the control roots, indicating that expression of RiPho4 was effectively knocked down  conditions. As a result, the P concentration in AM plants of RiPho4-silenced lines were significantly reduced compared with control plants  when compared with control roots  assay. It is known that the common binding motif of Pho4 transcription factor is CACGTG/T (cis-regulatory elements) in the promoters of three Pi transporter genes RiPT1, RiPT2, and RiPT3 for further studies  were strongly inhibited under such conditions . Therefore, the transcription factor RiPho4 can directly regulate the Pi transporter genes RiPT1, RiPT2 and RiPT3 in PHO pathway. Collectively, the Y1H results implicate that RiPho4 may act as a transcriptional activator in the PHO pathway in R. irregularis.As a result, the Y1HGold yeast cells containing pAbAi vector with Pvely see . In the nditions . These dR. irregularis mediating Pi uptake and homeostasis in AM symbiosis of E. grandis by investigating the expression, localization and function of RiPho4, a transcription factor of the PHO pathway.Recent years, more researchers have reported the physiological responses of Eucalyptus species to low Pi stress , and focR. irregularis-E. grandis interaction in this study  transcription factor to activate the expression of PHO downstream genes in response to Pi limitation  from N. crassa is considered to be a factor to activate the transcription of Pi transporter genes , it is predicted that RiPho4 may also have regulatory functions on these Pi and Poly-P metabolism genes , the Key Projects of Guangzhou of Science and Technology Plan (grant no. 201904020022), and the Laboratory of Lingnan Modern Agriculture Project (NZ2021025), the Guangdong Basic and Applied Basic Research Foundation (grant no. 2022A1515012013).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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