A rapid, equipment-free method for detecting avirulence genes of Pyricularia oryzae using a lateral flow strip-based RPA assay.

Rice blast, caused by Pyricularia oryzae, is one of the most destructive rice diseases worldwide. Using resistant rice varieties is the most cost-effective way to control rice blast. Consequently, it is critical to monitor the distribution frequency of avirulence genes in rice planting field to facilitate the breedings of resistant rice varieties. In this study, we established a rapid RPA-LFD detection system for the identification of AvrPik, Avr-Piz-t and Avr-Pi9. The optimized reaction temperature and duration were 37°C and 20 min, indicating that the reaction system could be initiated by body temperature without relying on any precision instruments. Specificity analysis showed that the primer and probe combinations targeting three Avr genes exhibited a remarkable specificity for at genus-level detection. Under the optimized condition, the lower detected thresholds of AvrPik, Avr-Piz-t and Avr-Pi9 were 10 fg/µl, 100 fg/µl and 10 pg/µl, respectively. Notably, the detection sensitivity of three Avr genes was much higher than that of PCR. In addition, we also successfully detected the presence of AvrPik, Avr-Piz-t and Avr-Pi9 in the leaf and panicle blast lesions with the RPA-LFD detection system. In particular, the genomic DNA was extracted using the simpler PEG-NaOH rapid extraction method. In summary, we developed the RPA detection system for AvrPik, Avr-Pi9 and Avr-Piz-t, combined with the PEG-NaOH rapid DNA extraction method. The innovative approach achieved rapid, real-time and accurate detection of three Avr genes in the field, which is helpful to understand the distribution frequency of the three Avr genes in the field and provide theoretical reference for the scientific layout of rice resistant varieties.

The Ras GTPase-activating protein UvGap1 orchestrates conidiogenesis and pathogenesis in the rice false smut fungus Ustilaginoidea virens.

Ras GTPase-activating proteins (Ras GAPs) act as negative regulators for Ras proteins and are involved in various signalling processes that influence cellular functions. Here, the function of four Ras GAPs, UvGap1 to UvGap4, was identified and analysed in Ustilaginoidea virens, the causal agent of rice false smut disease. Disruption of UvGAP1 or UvGAP2 resulted in reduced mycelial growth and an increased percentage of larger or dumbbell-shaped conidia. Notably, the mutant ΔUvgap1 completely lost its pathogenicity. Compared to the wild-type strain, the mutants ΔUvgap1, ΔUvgap2 and ΔUvgap3 exhibited reduced tolerance to H2 O2 oxidative stress. In particular, the ΔUvgap1 mutant was barely able to grow on the H2 O2 plate, and UvGAP1 was found to influence the expression level of genes involved in reactive oxygen species synthesis and scavenging. The intracellular cAMP level in the ΔUvgap1 mutant was elevated, as UvGap1 plays an important role in maintaining the intracellular cAMP level by affecting the expression of phosphodiesterases, which are linked to cAMP degradation in U. virens. In a yeast two-hybrid assay, UvRas1 and UvRasGef (Ras guanyl nucleotide exchange factor) physically interacted with UvGap1. UvRas2 was identified as an interacting partner of UvGap1 through a bimolecular fluorescence complementation assay and affinity capture-mass spectrometry analysis. Taken together, these findings suggest that the UvGAP1-mediated Ras pathway is essential for the development and pathogenicity of U. virens.

UvVelC is important for conidiation and pathogenicity in the rice false smut pathogen Ustilaginoidea virens.

Rice false smut disease is one of the most significant rice diseases worldwide. Ustilaginoidea virens is the causative agent of this disease. Although several developmental and pathogenic genes have been identified and functionally analyzed, the pathogenic molecular mechanisms of U. virens remain elusive. The velvet family regulatory proteins are involved in fungal development, conidiation, and pathogenicity. In this study, we demonstrated the function of the VelC homolog UvVELC in U. virens. We identified the velvet family protein UvVELC and characterized its functions using a target gene deletion-strategy. Deletion of UvVELC resulted in conidiation failure and pathogenicity. The UvVELC expression levels during infection suggested that this gene might be involved in the early infection process. UvVELC is also important in resistance to abiotic stresses, the utilization efficiency of glucose, stachyose, raffinose, and other sugars, and the expression of transport-related genes. Moreover, UvVELC could physically interact with UvVEA in yeast, and UvVELC/UvVEA double-knockout mutants also failed in conidiation and pathogenicity. These results indicate that UvVELC play a critical role in the conidiation and pathogenicity in U. virens. Functional analysis indicated that UvVELC-mediated conidiation and nutrient acquisition from rice regulates the pathogenicity of U. virens. Understanding the function of the UvVELC homolog could provide a potential molecular target for controlling rice false smut disease.

Genome-Wide Prediction and Analysis of Oryza Species NRP Genes in Rice Blast Resistance.

Members of the N-rich proteins (NRPs) gene family play important roles in the plant endoplasmic reticulum stress in response, which can be triggered by plant pathogens' infection. Previous studies of the NRP gene family have been limited to only a few plants, such as soybean and Arabidopsis thaliana. Thus, their evolutionary characteristics in the Oryza species and biological functions in rice defense against the pathogenic fungus Magnaporthe oryzae have remained unexplored. In the present study, we demonstrated that the NRP genes family may have originated in the early stages of plant evolution, and that they have been strongly conserved during the evolution of the Oryza species. Domain organization of NRPs was found to be highly conserved within but not between subgroups. OsNRP1, an NRP gene in the Oryza sativa japonica group, was specifically up-regulated during the early stages of rice-M. oryzae interactions-inhibited M. oryzae infection. Predicted protein-protein interaction networks and transcription-factor binding sites revealed a candidate interactor, bZIP50, which may be involved in OsNRP1-mediated rice resistance against M. oryzae infection. Taken together, our results established a basis for future studies of the NRP gene family and provided molecular insights into rice immune responses to M. oryzae.

The Adaptor Protein UvSte50 Governs Fungal Pathogenicity of Ustilaginoidea virens via the MAPK Signaling Pathway.

The mitogen-activated protein kinase (MAPK) signaling pathways regulate diverse cellular processes and have been partially characterized in the rice false smut fungus Ustilaginoidea virens. UvSte50 has been identified as a homolog to Saccharomyces cerevisiae Ste50, which is known to be an adaptor protein for MAPK cascades. ΔUvste50 was found to be defective in conidiation, sensitive to hyperosmotic and oxidative stresses, and non-pathogenic. The mycelial expansion of ΔUvste50 inside spikelets of rice terminated at stamen filaments, eventually resulting in a lack of formation of false smut balls on spikelets. We determined that UvSte50 directly interacts with both UvSte7 (MAPK kinase; MEK) and UvSte11 (MAPK kinase kinase; MEKK), where the Ras-association (RA) domain of UvSte50 is indispensable for its interaction with UvSte7. UvSte50 also interacts with UvHog1, a MAP kinase of the Hog1-MAPK pathway, which is known to have important roles in hyphal growth and stress responses in U. virens. In addition, affinity capture-mass spectrometry analysis and yeast two-hybrid assay were conducted, through which we identified the interactions of UvSte50 with UvRas2, UvAc1 (adenylate cyclase), and UvCap1 (cyclase-associated protein), key components of the Ras/cAMP signaling pathway in U. virens. Together, UvSte50 functions as an adaptor protein interacting with multiple components of the MAPK and Ras/cAMP signaling pathways, thus playing critical role in plant infection by U. virens.

A potato late blight resistance gene protects against multiple Phytophthora species by recognizing a broadly conserved RXLR-WY effector.

Species of the genus Phytophthora, the plant killer, cause disease and reduce yields in many crop plants. Although many Resistance to Phytophthora infestans (Rpi) genes effective against potato late blight have been cloned, few have been cloned against other Phytophthora species. Most Rpi genes encode nucleotide-binding domain, leucine-rich repeat-containing (NLR) immune receptor proteins that recognize RXLR (Arg-X-Leu-Arg) effectors. However, whether NLR proteins can recognize RXLR effectors from multiple Phytophthora species has rarely been investigated. Here, we identified a new RXLR-WY effector AVRamr3 from P. infestans that is recognized by Rpi-amr3 from a wild Solanaceae species Solanum americanum. Rpi-amr3 associates with AVRamr3 in planta. AVRamr3 is broadly conserved in many different Phytophthora species, and the recognition of AVRamr3 homologs by Rpi-amr3 activates resistance against multiple Phytophthora pathogens, including the tobacco black shank disease and cacao black pod disease pathogens P. parasitica and P. palmivora. Rpi-amr3 is thus the first characterized resistance gene that acts against P. parasitica or P. palmivora. These findings suggest a novel path to redeploy known R genes against different important plant pathogens.

The Velvet Protein UvVEA Regulates Conidiation and Chlamydospore Formation in Ustilaginoidea virens.

Rice false smut, caused by Ustilaginoidea virens, is a serious disease of rice worldwide, severely reducing the quantity and quality of rice production. The conserved fungal velvet proteins are global regulators of diverse cellular processes. We identified and functionally characterized two velvet genes, UvVEA and UvVELB, in U. virens. The deletion of these genes affected the conidiation of U. virens but had no effect on the virulence of this pathogen. Interestingly, the ΔUvVEA mutants appeared in the form of smaller false smut balls with a reduced number of chlamydospores compared with the wide-type strains. In addition, the deletion of UvVEA affected the expression of some transmembrane transport genes during chlamydospore formation and rice false smut balls development. Furthermore, the ΔUvVEA mutants were shown to be defective in the utilization of glucose. These findings proved the regulatory mechanism underlying the formation of rice false smut balls and chlamydospores and provided a basis for the further exploration of the mechanism of these processes.

Identification of Differentially Expressed Genes Reveal Conserved Mechanisms in the Rice-Magnaporthe oryzae Interaction.

Magnaporthe oryzae causes rice blast disease and is responsible for major losses in rice production worldwide. Although numerous studies have focused on the interactions between Oryza sativa and M. oryzae, to date, the conserved mechanisms remain in part unclear. In this study, a comparative analysis of transcriptomes of O. sativa L. ssp. japonica cv. 'Nipponbare' interacting with three M. oryzae strains (248, 235, and 163) were performed to explore the conserved molecular mechanisms. Differentially expressed genes with similar expression patterns in the interactions between cultivar 'Nipponbare' and three M. oryzae strains were defined as Conserved Differentially Expressed Genes (CDEGs). These included 3,647 O. sativa CDEGs and 3,655 M. oryzae CDEGs. Four rice CDEGs (LOC_Os03g19270, LOC_Os07g36600, LOC_Os05g28740, and LOC_Os01g32780) encoding universal stress protein (USP) were induced within 24 h post-inoculation (hpi) by three M. oryzae strains. Meanwhile, overexpression of LOC_Os07g36600 resulted in enhanced rice resistance against M. oryzae. Furthermore, four rice genes coding light-harvesting chlorophyll a/b-binding (LHC) protein (LOC_Os02g52650, LOC_Os09g12540, LOC_Os11g13850, LOC_Os05g22730) were also identified as CDEGs and were induced at 48 hpi, which might contribute to blast resistance through reactive oxygen species (ROS) accumulation. MoCDIP4 is M. oryzae effector inducing rice cell death and were verified that include AA9 CAZy domain (namely GH61 domain). In this study, we found seven MoCDIP4-homologous genes coding proteins with signal peptides and AA9 CAZy domains, which were continuously up-regulated across all infection stages relative to uninoculated control. This study uncovered that genes are required for conserved mechanisms of rice-M. oryzae interaction, which includes rice genes encoding USP proteins and LHC proteins, as well as M. oryzae genes encoding AA9 proteins. This study will help us to understand how O. sativa responds to M. oryzae infections and the molecular mechanisms of M. oryzae pathogenicity.

Autophagy-related protein UvAtg7 contributes to mycelial growth, virulence, asexual reproduction and cell stress response in rice false smut fungus Ustilaginoidea virens.

Autophagy is a conserved mechanism for nutrient and cytoplasmic components recycling in eukaryotic cell, in which E1-like enzyme Atg7 activates ubiquitin-like conjugation in the autophagy pathway. In plant pathogenic fungi Ustilaginoidea virens, UvAtg7, an ortholog of AAtg7 in baker's yeast was identified and functionally investigated. UvAtg7 was confirmed to be essential for autophagy, because the disruption of UvATG7 gene in U. virens completely blocked the fusion of autophagosome-like into vacuoles and catalytic degradation of GFP-UvAtg8 under N-starving condition. The fluorescent signal indicated UvAtg7 protein was dispersed in cytoplasma, but spatially coordinated with core autophagy protein UvAtg8 on occasion. Interestingly, disruption of UvATG7 in U. virens caused slightly reduction in mycelial growth, but resulted in a considerable decrease in virulence, conidia production in YT broth and chlamydospore formation on rice false smut balls. Moreover, the UvATG7 deletion mutants exhibited increased sensitivity to cell wall integrity stress caused by congo red and calcofluor white, meanwhile the UvATG7 deletion mutants showed decreased sensitivity to osmotic stress, cell membrane stress and reactiveoxygen stress caused by sorbitol, sodium dodecyl sulfate and H2O2, respectively. All of these defects in UvATG7 deletion mutants could be partially or completely restored by gene complementation. In general, our study indicates that UvAtg7 is essential in autophagy pathway and contributes to mycelial growth, virulence, asexual reproduction and cell stress response in U. virens.

Loop-Mediated Isothermal Amplification for Rapid Detection of Mating Types of Villosiclava virens.

Rice false smut (RFS), caused by Villosiclava virens, is an important fungal disease in panicles of rice. V. virens is a heterothallic ascomycete controlled by two opposite idiomorphs, MAT1-1 and MAT1-2. Previous study showed that sexual reproduction of V. virens plays an important role in the epidemic of RFS. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay to detect the mating type of V. virens easily and rapidly by using specific primers based on the mating type genes MAT1-1-2 and MAT1-2-1, respectively. The LAMP assay used only a water/dry bath and could recognize the mating type of V. virens in just 45 min. The LAMP assay was so sensitive that it could detect small amounts of V. virens genomic DNA (as low as 2.0 pg of MAT1-1 and 200.0 pg of MAT1-2) and was 10 times more sensitive than PCR. In addition, we demonstrated the application of mating type via LAMP assay by assessing the genomic DNA of V. virens isolated from rice fields. The high efficiency and specificity of this LAMP assay suggest that it can be used as a rapid testing tool in mating type recognition of V. virens isolates in the field.

SUN-Family Protein UvSUN1 Regulates the Development and Virulence of Ustilaginoidea virens.

Ustilaginoidea virens, the causal agent of rice false smut disease, is an important plant pathogen that causes severe quantitative and qualitative losses in rice worldwide. UvSUN1 is the only member of Group-I SUN family proteins in U. virens. In this work, the role of UvSUN1 in different aspects of the U. virens biology was studied by phenotypic analysis of Uvsun1 knockout strains. We identified that UvSUN1 was expressed during both conidial germination and the infection of rice. Disruption of the Uvsun1 gene affected the hyphal growth, conidiation, morphology of hyphae and conidia, adhesion and virulence. We also found that UvSUN1 is involved in the production of toxic compounds, which are able to inhibit elongation of the germinated seeds. Moreover, RNA-seq data showed that knockout of Uvsun1 resulted in misregulation of a subset of genes involved in signal recognition and transduction system, glycometabolism, cell wall integrity, and secondary metabolism. Collectively, this study reveals that Uvsun1 is required for growth, cell wall integrity and pathogenicity of U. virens, thereby providing new insights into the function of SUN family proteins in the growth and pathogenesis of this pathogen.

Pyricularia sp. jiangsuensis, a new cryptic rice panicle blast pathogen from rice fields in Jiangsu Province, China.

Pyricularia oryzae is a multi-host pathogen causing cereal disease, including the devastating rice blast. Panicle blast is a serious stage, leading to severe yield loss. Thirty-one isolates (average 4.1%) were collected from the rice panicle lesions at nine locations covering Jiangsu province from 2010 to 2017. These isolates were characterized as Pyricularia sp. jiangsuensis distinct from known Pyricularia species. The representative strain 18-2 can infect rice panicle, root and five kinds of grasses. Intriguingly, strain 18-2 can co-infect rice leaf with P. oryzae Guy11. The whole genome of P. sp. jiangsuensis 18-2 was sequenced. Nine effectors were distributed in translocation or inversion region, which may link to the rapid evolution of effectors. Twenty-one homologues of known blast-effectors were identified in strain 18-2, seven effectors including the homologues of SLP1, BAS2, BAS113, CDIP2/3, MoHEG16 and Avr-Pi54, were upregulated in the sample of inoculated panicle with strain 18-2 at 24 hpi compared with inoculation at 8 hpi. Our results provide evidences that P. sp. jiangsuensis represents an addition to the mycobiota of blast disease. This study advances our understanding of the pathogenicity of P. sp. jiangsuensis to hosts, which sheds new light on the adaptability in the co-evolution of pathogen and host.

The N-terminus of an Ustilaginoidea virens Ser-Thr-rich glycosylphosphatidylinositol-anchored protein elicits plant immunity as a MAMP.

Many pathogens infect hosts through specific organs, such as Ustilaginoidea virens, which infects rice panicles. Here, we show that a microbe-associated molecular pattern (MAMP), Ser-Thr-rich Glycosyl-phosphatidyl-inositol-anchored protein (SGP1) from U. virens, induces immune responses in rice leaves but not panicles. SGP1 is widely distributed among fungi and acts as a proteinaceous, thermostable elicitor of BAK1-dependent defense responses in N. benthamiana. Plants specifically recognize a 22 amino acid peptide (SGP1 N terminus peptide 22, SNP22) in its N-terminus that induces cell death, oxidative burst, and defense-related gene expression. Exposure to SNP22 enhances rice immunity signaling and resistance to infection by multiple fungal and bacterial pathogens. Interestingly, while SGP1 can activate immune responses in leaves, SGP1 is required for U. virens infection of rice panicles in vivo, showing it contributes to the virulence of a panicle adapted pathogen.

A Phytophthora sojae CRN effector mediates phosphorylation and degradation of plant aquaporin proteins to suppress host immune signaling.

Phytophthora genomes encode a myriad of Crinkler (CRN) effectors, some of which contain putative kinase domains. Little is known about the host targets of these kinase-domain-containing CRNs and their infection-promoting mechanisms. Here, we report the host target and functional mechanism of a conserved kinase CRN effector named CRN78 in a notorious oomycete pathogen, Phytophthora sojae. CRN78 promotes Phytophthora capsici infection in Nicotiana benthamiana and enhances P. sojae virulence on the host plant Glycine max by inhibiting plant H2O2 accumulation and immunity-related gene expression. Further investigation reveals that CRN78 interacts with PIP2-family aquaporin proteins including NbPIP2;2 from N. benthamiana and GmPIP2-13 from soybean on the plant plasma membrane, and membrane localization is necessary for virulence of CRN78. Next, CRN78 promotes phosphorylation of NbPIP2;2 or GmPIP2-13 using its kinase domain in vivo, leading to their subsequent protein degradation in a 26S-dependent pathway. Our data also demonstrates that NbPIP2;2 acts as a H2O2 transporter to positively regulate plant immunity and reactive oxygen species (ROS) accumulation. Phylogenetic analysis suggests that the phosphorylation sites of PIP2 proteins and the kinase domains of CRN78 homologs are highly conserved among higher plants and oomycete pathogens, respectively. Therefore, this study elucidates a conserved and novel pathway used by effector proteins to inhibit host cellular defenses by targeting and hijacking phosphorylation of plant aquaporin proteins.

A complex resistance locus in Solanum americanum recognizes a conserved Phytophthora effector.

Late blight caused by Phytophthora infestans greatly constrains potato production. Many Resistance (R) genes were cloned from wild Solanum species and/or introduced into potato cultivars by breeding. However, individual R genes have been overcome by P. infestans evolution; durable resistance remains elusive. We positionally cloned a new R gene, Rpi-amr1, from Solanum americanum, that encodes an NRC helper-dependent CC-NLR protein. Rpi-amr1 confers resistance in potato to all 19 P. infestans isolates tested. Using association genomics and long-read RenSeq, we defined eight additional Rpi-amr1 alleles from different S. americanum and related species. Despite only ~90% identity between Rpi-amr1 proteins, all confer late blight resistance but differentially recognize Avramr1 orthologues and paralogues. We propose that Rpi-amr1 gene family diversity assists detection of diverse paralogues and alleles of the recognized effector, facilitating durable resistance against P. infestans.

Dissection of the general two-step di-C-glycosylation pathway for the biosynthesis of (iso)schaftosides in higher plants.

Schaftoside and isoschaftoside are bioactive natural products widely distributed in higher plants including cereal crops and medicinal herbs. Their biosynthesis may be related with plant defense. However, little is known on the glycosylation biosynthetic pathway of these flavonoid di-C-glycosides with different sugar residues. Herein, we report that the biosynthesis of (iso)schaftosides is sequentially catalyzed by two C-glycosyltransferases (CGTs), i.e., CGTa for C-glucosylation of the 2-hydroxyflavanone aglycone and CGTb for C-arabinosylation of the mono-C-glucoside. The two enzymes of the same plant exhibit high homology but remarkably different sugar acceptor and donor selectivities. A total of 14 CGTa and CGTb enzymes were cloned and characterized from seven dicot and monocot plants, including Scutellaria baicalensis, Glycyrrhiza uralensis, Oryza sativa ssp. japonica, and Zea mays, and the in vivo functions for three enzymes were verified by RNA interference and overexpression. Through transcriptome analysis, we found homologous genes in 119 other plants, indicating this pathway is general for the biosynthesis of (iso)schaftosides. Furthermore, we resolved the crystal structures of five CGTs and realized the functional switch of SbCGTb to SbCGTa by structural analysis and mutagenesis of key amino acids. The CGT enzymes discovered in this paper allow efficient synthesis of (iso)schaftosides, and the general glycosylation pathway presents a platform to study the chemical defense mechanisms of higher plants.

Identification of Avramr1 from Phytophthora infestans using long read and cDNA pathogen-enrichment sequencing (PenSeq).

Potato late blight, caused by the oomycete pathogen Phytophthora infestans, significantly hampers potato production. Recently, a new Resistance to Phytophthora infestans (Rpi) gene, Rpi-amr1, was cloned from a wild Solanum species, Solanum americanum. Identification of the corresponding recognized effector (Avirulence or Avr) genes from P. infestans is key to elucidating their naturally occurring sequence variation, which in turn informs the potential durability of the cognate late blight resistance. To identify the P. infestans effector recognized by Rpi-amr1, we screened available RXLR effector libraries and used long read and cDNA pathogen-enrichment sequencing (PenSeq) on four P. infestans isolates to explore the untested effectors. Using single-molecule real-time sequencing (SMRT) and cDNA PenSeq, we identified 47 highly expressed effectors from P. infestans, including PITG_07569, which triggers a highly specific cell death response when transiently coexpressed with Rpi-amr1 in Nicotiana benthamiana, suggesting that PITG_07569 is Avramr1. Here we demonstrate that long read and cDNA PenSeq enables the identification of full-length RXLR effector families and their expression profile. This study has revealed key insights into the evolution and polymorphism of a complex RXLR effector family that is associated with the recognition by Rpi-amr1.

MAT1-1-3, a Mating Type Gene in the Villosiclava virens, Is Required for Fruiting Bodies and Sclerotia Formation, Asexual Development and Pathogenicity.

Villosiclava virens is the prevalent causative pathogen of rice false smut, a destructive rice disease. Mating-type genes play a vital role in the evolution of mating systems in fungi. Some fungi have lost MAT1-1-3, one of the mating-type genes, during evolution, whereas others still retain MAT1-1-3. However, how MAT1-1-3 regulates the sexual development of heterothallic V. virens remains unknown. Here, we generated the MAT1-1-3 mutants, which exhibited defects in vegetative growth, stress response, pathogenicity, sclerotia formation and fruiting body maturation. An artificial outcrossing inoculation assay showed that the Δmat1-1-3 mutant was unable to produce sclerotia. Unexpectedly, the Δmat1-1-3 mutant could form immature fruiting bodies without mating on potato sucrose agar medium (PSA) compared with the wild-type strain, most likely by activating the truncated MAT1-2-1 transcription to regulate the sexual development. Moreover, RNA-seq data showed that knockout of MAT1-1-3 results in misregulation of a subset of genes involved in sexual development, MAPK signaling, cell wall integrity, autophagy, epigenetic modification, and transcriptional regulation. Collectively, this study reveals that MAT1-1-3 is required for asexual and sexual development, and pathogenicity of V. virens, thereby provides new insights into the function of mating-type genes in the fungi life cycle and infection process.

Two mating-type genes MAT1-1-1 and MAT1-1-2 with significant functions in conidiation, stress response, sexual development, and pathogenicity of rice false smut fungus Villosiclava virens.

Rice false smut caused by Villosiclava virens is one of the destructive diseases on panicles of rice. Sexual development of V. virens, controlled by mating-type locus, plays an important role in the prevalence of rice false smut and genetic diversity of the pathogen. However, how the mating-type genes mediate sexual development of the V. virens remains largely unknown. In this study, we characterized the two mating-type genes, MAT1-1-1 and MAT1-1-2, in V. virens. MAT1-1-1 knockout mutant showed defects in hyphal growth, conidia morphogenesis, sexual development, and increase in the tolerance to salt and osmotic stress. Targeted deletion of MAT1-1-2 not only impaired the sclerotia formation and pathogenicity of V. virens, but also reduced the production of conidia. The MAT1-1-2 mutant showed increases in tolerance to salt and hydrogen peroxide stress, but decreases in tolerance to osmotic stress. Yeast two-hybrid assay showed that MAT1-1-1 interacted with MAT1-1-2, indicating that those proteins might form a complex to regulate sexual development. In addition, MAT1-1-1 localized in the nucleus, and MAT1-1-2 localized in the cytoplasm. Collectively, our results demonstrate that MAT1-1-1 and MAT1-1-2 play important roles in the conidiation, stress response, sexual development, and pathogenicity of V. virens, thus providing new insights into the function of mating-type gene.

A Homeobox Transcription Factor UvHOX2 Regulates Chlamydospore Formation, Conidiogenesis, and Pathogenicity in Ustilaginoidea virens.

Rice false smut fungus (teleomorph: Villosiclava virens; anamorph: Ustilaginoidea virens) can generate chlamydospores and survive winter under field conditions. The chlamydospore is considered as an important infection source of the disease. However, little is known about the regulatory mechanism of the chlamydospore production. In this study, we identified a defective homeobox transcription factor (designated as UvHOX2) gene in a U. virens random insertional mutant B-766 that could not form chlamydospores. To confirm the regulatory function of UvHOX2, an Agrobacterium tumefaciens mediated transformation- and CRISPR/Cas9- based targeted gene replacement method was developed. The UvHox2 deletion mutants completely failed to produce chlamydospores, showed reduced conidia production and decreased virulence, and was hyper-sensitive to oxidative, osmotic, and cell wall stresses. We confirmed that UvHOX2 is located in the nuclei of U. virens, and the expression of UvHox2 was the strongest during the early stage of chlamydospore and conidium formation. Global transcription pattern of UvHOX2 was also determined by RNA-seq in this study, and several genes that might be down-stream of UvHOX2 regulation were identified. The results will better our understanding of the molecular mechanism of chlamydospore formation in U. virens as a model fungus.