Search

Plants perceive pathogens and induce robust transcriptional reprogramming to rapidly achieve immunity. The mechanisms of how immune-related genes are transcriptionally regulated remain largely unknown. Previously, the trihelix transcriptional factor ARABIDOPSIS SH4-RELATED 3 (ASR3) was shown to negatively regulate pattern-triggered immunity (PTI) in Arabidopsis thaliana. Here, we identified another trihelix family member ASR3-Interacting Transcriptional Factor 1 (AITF1) as an interacting protein of ASR3. ASR3-Interacting Transcriptional Factor 1 and ASR3 form heterogenous and homogenous dimers in planta. Both aitf1 and asr3 single mutants exhibited increased resistance against the bacterial pathogen Pseudomonas syringae, but the double mutant showed reduced resistance, suggesting AITF1 and ASR3 interdependently regulate immune gene expression and resistance. Overexpression of AITF1 triggered autoimmunity dependently on its DNA-binding ability and the presence of ASR3. Notably, autoimmunity caused by overexpression of AITF1 was dependent on a TIR-NBS-LRR (TNL) protein suppressor of AITF1-induced autoimmunity 1 (SAA1), as well as enhanced disease susceptibility 1 (EDS1), the central regulator of TNL signaling. ASR3-Interacting Transcriptional Factor 1 and ASR3 directly activated SAA1 expression through binding to the GT-boxes in SAA1 promoter. Collectively, our results revealed a mechanism of trihelix transcription factor complex in regulating immune gene expression, thereby modulating plant disease resistance and autoimmunity.

Sclerotinia sclerotiorum is a well-known phytopathogenic fungus with a wide host range. Identifying novel mycoviruses in phytopathogenic fungi is necessary to develop novel strategies for plant health protection and contribute to understanding the origin of viruses. Six new mycoviruses with positive single-stranded RNA genomes co-infecting the hypovirulent strain SCH733 of S. sclerotiorum were identified using a metatranscriptomic approach, and their complete genome sequences were molecularly determined. These mycoviruses belong to the following five families: Narnaviridae, Mitoviridae, Deltaflexviridae, Botourmiaviridae, and Ambiguiviridae. Three of these mycoviruses belong to existing International Committee on Taxonomy of Viruses (ICTV)-recognized species. Two of these newly identified mycoviruses have unique genomic features that are significantly different from those of all known mycoviruses. Phylogenetic analysis revealed that these six mycoviruses included close as well as distant relatives of known mycoviruses, thereby providing new insight into virus evolution and classification. Mycovirus horizontal transmission and elimination experiments revealed that Sclerotinia sclerotiorum narnavirus 5 is associated with hypovirulence of S. sclerotiorum, although we have not shown that it is independently responsible for the hypovirulence phenotype. This study broadens the diversity of known mycoviruses infecting S. sclerotiorum and provides a clue toward limiting hypovirulence in S. sclerotiorum.

A new positive-sense single-stranded RNA (+ssRNA) mycovirus, Verticillium dahliae magoulivirus 1 (VdMoV1), was isolated from two strains (2-19 and XLZ70) of Verticillium dahliae. The complete genome of VdMoV1 is 2303 nucleotides (nt) in length and has a large open reading frame (nt positions from 61 to 1938) encoding an RNA-dependent RNA polymerase (RdRp). A multiple sequence alignment indicated that the central region of the RdRp encoded by VdMoV1 contains eight typical viral RdRp motifs. BLASTp analysis demonstrated that VdMoV1 has the highest sequence identity (86.88%) to Bremia lactucae associated ourmia-like virus 2 (BlaOLV2). Phylogenetic analysis revealed that VdMoV1 is a new member of the genus Magoulivirus. As far as we know, VdMoV1 is the first reported member of the family Botourmiaviridae infecting V. dahliae.

Plants recognize microbe-associated molecular patterns (MAMPs) to activate immune responses and defense priming to defend against pathogen infections. Transcriptional regulation of gene expression is crucial for plant immunity and is mediated by multiple factors, including DNA methylation. However, it remains unknown whether and how DNA demethylation contributes to immune responses in MAMP-triggered immunity. Here, we report that active DNA demethylation is required for MAMP-triggered immunity to bacterial pathogens. The rdd-2 triple mutant carrying mutations in ROS1, DML2, and DML3 that encode DNA glycosylases, which are key DNA demethylation enzymes, exhibits compromised immune responses triggered by the MAMPs flg22 and elf18. Genome-wide methylome analysis reveals that flg22 induces rapid and specific DNA demethylation in an RDD-dependent manner. The expression levels of salicylic acid signaling-related and phytoalexin biosynthesis-related genes are tightly associated with the flg22-induced promoter demethylation. The compromised accumulation of priming compounds and antimicrobial metabolites ultimately leads to a defense priming defect in the rdd-2 mutant. Our results reveal the critical role of active DNA demethylation in the MAMP-triggered immune response and provide unique insight into the molecular mechanism of flg22-modulated DNA demethylation.

A significant number of mycoviruses have been identified that are related to plant viruses, but their evolutionary relationships are largely unexplored. A fusarivirus, Rhizoctonia solani fusarivirus 4 (RsFV4), was identified in phytopathogenic fungus Rhizoctonia solani (R. solani) strain XY74 co-infected by an alphaendornavirus. RsFV4 had a genome of 10,833 ​nt (excluding the poly-A tail), and consisted of four non-overlapping open reading frames (ORFs). ORF1 encodes an 825 aa protein containing a conserved helicase domain (Hel1). ORF3 encodes 1550 aa protein with two conserved domains, namely an RNA-dependent RNA polymerase (RdRp) and another helicase (Hel2). The ORF2 and ORF4 likely encode two hypothetical proteins (520 and 542 aa) with unknown functions. The phylogenetic analysis based on Hel2 and RdRp suggest that RsFV4 was positioned within the fusarivirus group, but formed an independent branch with three previously reported fusariviruses of R. solani. Notably, the Hel1 and its relatives were phylogenetically closer to helicases of potyviruses and hypoviruses than fusariviruses, suggesting fusarivirus Hel1 formed an evolutionary link between these three virus groups. This finding provides evidence of the occurrence of a horizontal gene transfer or recombination event between mycoviruses and plant viruses or between mycoviruses. Our findings are likely to enhance the understanding of virus evolution and diversity.

Ralstonia solanacearum is a devastating soil-borne bacterial pathogen capable of infecting many plant species, including tomato (Solanum lycopersicum). However, the perception of Ralstonia by the tomato immune system and the pathogen's counter-defense strategy remain largely unknown. Here, we show that PehC, a specific exo-polygalacturonase secreted by Ralstonia, acts as an elicitor that triggers typical immune responses in tomato and other Solanaceous plants. The elicitor activity of PehC depends on its N-terminal epitope, and not on its polygalacturonase activity. The recognition of PehC specifically occurs in tomato roots and relies on unknown receptor-like kinase(s). Moreover, PehC hydrolyzes plant pectin-derived oligogalacturonic acids (OGs), a type of damage-associated molecular pattern (DAMP), which leads to the release of galacturonic acid (GalA), thereby dampening DAMP-triggered immunity (DTI). Ralstonia depends on PehC for its growth and early infection and can utilize GalA as a carbon source in the xylem. Our findings demonstrate the specialized and dual functions of Ralstonia PehC, which enhance virulence by degrading DAMPs to evade DTI and produce nutrients, a strategy used by pathogens to attenuate plant immunity. Solanaceous plants have evolved to recognize PehC and induce immune responses, which highlights the significance of PehC. Overall, this study provides insight into the arms race between plants and pathogens.

Kiwi is a popular fruit consumed worldwide. A number of fungal pathogens have been reported to cause postharvest rot of kiwifruit, and Botryosphaeriaceae species are the major causal agents of the disease. In this study, 18 isolates belonged to the genus Neofusicoccum (family Botryosphaeriaceae) were isolated from 247 symptomatic kiwifruits of the cultivars Jinyan, Jintao, and Jinkui collected from orchards in Hubei and Jiangxi Provinces, China. Among the isolates, three grouped with various known N. parvum isolates, whereas the remaining 15 formed two independent clades. On the basis of further phylogenetic analyses with concatenated sequences of ITS and three genes encoding translation elongation factor 1-alpha (TEF), β-tubulin (TUB), and DNA-dependent RNA polymerase II subunit (RPB2), as well as morphological characteristics, two new species, N. actinidiae and N. guttata, were proposed. Their pathogenicity to kiwi, apple, and citrus fruits was also confirmed.

Fungal segmented negative-sense RNA viruses (SNSRVs) have been frequently found. Only the large segment encoding RNA-dependent RNA polymerase (RdRp) has been reported in most fungal SNSRVs, except for a few fungal SNSRVs reported to encode nucleocapsids, nonstructural proteins, or movement proteins.

Sclerotinia sclerotiorum reovirus 1 (SsReV1) was previously reported to infect hypovirulent strain SCH941 of the phytopathogenic fungus Sclerotinia sclerotiorum and to contain 11 double-stranded RNA (dsRNA) segments (S1-S11). Here, we report that SsReV1 is actually composed of 12 dsRNA segments instead of 11. The full-length nucleotide sequence of the twelfth segment (S12) was determined using a combination of RACE and high-throughput sequencing methods. S12 is 1217 nucleotides in length and has highly conserved terminal sequences that resemble those of the other 11 segments of SsReV1. S12 contains a single open reading frame encoding a protein (VP12) of 311 amino acids. Although regular BLAST analysis did not reveal any similarity of VP12 to known sequences, it was found to be homologous to the VP11 of Colorado tick fever virus of the genus Coltivirus when a hidden-Markov-model-based HHpred analysis was performed. A single-protoplast regeneration experiment suggested that S12 and S2 were maintained or lost in parallel. In summary, the SsReV1 genome consists of 12 dsRNA segments.

Long noncoding RNAs (lncRNAs) are crucial regulators of gene expression in many biological processes, but their biological functions remain largely unknown, especially in fungi. Fusarium graminearum is an important pathogen that causes the destructive disease Fusarium head blight (FHB) or head scab disease on wheat and barley. In our previous RNA sequencing (RNA‐Seq) study, we discovered that lncRsp1 is an lncRNA that is located +99 bp upstream of a putative sugar transporter gene, Fgsp1, with the same transcription direction. Functional studies revealed that ΔlncRsp1 and ΔFgsp1 were normal in growth and conidiation but had defects in ascospore discharge and virulence on wheat coleoptiles. Moreover, lncRsp1 and Fgsp1 were shown to negatively regulate the expression of several deoxynivalenol (DON) biosynthesis genes, TRI4, TRI5, TRI6, and TRI13, as well as DON production. Further analysis showed that the overexpression of lncRsp1 enhanced the ability of ascospore release and increased the mRNA expression level of the Fgsp1 gene, while lncRsp1‐silenced strains reduced ascospore discharge and inhibited Fgsp1 expression during the sexual reproduction stage. In addition, the lncRsp1 complementary strains lncRsp1‐LC‐1 and lncRsp1‐LC‐2 restored ascospore discharge to the level of the wild‐type strain PH‐1. Taken together, our results reveal the distinct and specific functions of lncRsp1 and Fgsp1 in F. graminearum and principally demonstrate that lncRsp1 can affect the release of ascospores by regulating the expression of Fgsp1., lncRsp1, a long noncoding RNA located +99 bp upstream of a putative sugar transporter gene Fgsp1, can affect the release of ascospores by regulating the expression of Fgsp1 in Fusarium graminearum.

A novel double-stranded RNA virus was isolated and identified from Beauveria bassiana Vuillemin, derived from the muscardine cadaver of an Ostrinia furnacalis larva in China. The virus contains six dsRNAs, and each viral dsRNA contains only one open reading frame (ORF). As in other polymycoviruses, dsRNA1 encodes an RNA-dependent RNA polymerase (RdRp), dsRNA3 encodes a methyltransferase (MTR), and dsRNA4 encodes a proline-alanine-serine-rich protein. A BLASTp search revealed that the viral RdRp domain showed 79.43%, 79.04%, and 59.05% sequence identity to Beauveria bassiana polymycovirus 2 and 3 (BbPmV-2, BbPmV-3) and Magnaporthe oryzae polymycovirus 1 (MoPmV-1), respectively. Phylogenetic analysis based on RdRp sequences showed that the phylogenetically closest relatives of this virus are BbPmV-2, BbPmV-3, and MoPmV-1. This virus, along with previously ill-defined polymycoviruses (BbPmV-2 and BbPmV-3), appears to belong to an as-yet-unestablished species. The findings further suggest that the virus is a new member of the genus Polymycovirus within the family Polymycoviridae, and we have named it "Beauveria bassiana polymycovirus 4" (BbPmV-4). However, the sixth dsRNA is a defective RNA with the same sequence as that of dsRNA4 except for a deletion of 312 bp from nt 185 to nt 496, but it still contains a complete ORF. To our knowledge, this is the first report of the existence of a defective RNA in a polymycovirus.

Chitinase plays an important role in plant resistance against chitin containing pathogens by degrading chitin. Clubroot, caused by Plasmodiophora brassicae, is a major disease in cruciferous crops worldwide, including vegetable and rapeseed. Chitin is the main component of P. brassicae spore cell walls, chitinases can enhance the plant’s defense system as they act on chitin. However, the function of chitinases in P. brassicae has not reported. Here, we found that chitin is the functional component of P. brassicae by wheat germ agglutinin staining and commercial chitinase treatment . Chitin extracted from resting spores of P. brassicae was characterized by infrared spectroscopy, X-ray diffraction and thermo gravimetry analysis.The chitinase PbChia1 was identified by chitin pull-down assays combined with LC-MS/MS method. We also showed that PbChia1 was a typical secreted chitinase, could bind to chitin and showed chitinase activity in vitro, significantly degrade the number of resting spores of P. brassicae and significantly relieve the severity of clubroot symptom, the biocontrol effect was 61.29%. Overexpression of PbChia1 in Arabidopsis thaliana resulted in susceptibility to P. brassicae, increase host survival rate and seed yields, and transgenic plants enhanced in chitin-triggered reactive oxygen species burst, MAPK activation and expression of immune-related genes. PbChia1 transgenic plants showed resistance to other pathogens, such as biotrophic bacteria Pst DC3000, necrotrophic fungi Sclerotinia sclerotiorum 1980 and Rhizoctonia solani. The findings indicate that chitinases PbChia1 is a candidate gene with potential application for broad spectrum disease resistance in breeding plants to prevent clubroot.

Clubroot caused by the soil-borne protist pathogenPlasmodiophora brassicaeis one of the most devastating diseases of Brassica oil and vegetable crops worldwide. Understanding the pathogen infection strategy is crucial for the development of disease control. However, the molecular mechanism by which this pathogen promotes infection remains largely unknown. Here, we identified aP. brassicae-secreted effector PbE3-2 that impedes plant immunity by ubiquitinating the immune regulator RD21A for degradation. Overexpression ofPbE3-2inArabidopsis thalianaresulted in higher susceptibility toP. brassicaeand decreases in chitin-triggered reactive oxygen species burst and expression of marker genes in salicylic acid signaling. PbE3-2 interacted with and ubiquitinated RD21Ain vitroandin vivo. Mutant plants deficient inRD21Aexhibited similar susceptibility and compromsied immune responses as inPbE3-2overexpression plants. These results suggest thatP. brassicaepromotes clubroot disease through RD21A degradation mediated by the effector PbE3-2. As PbE3-2 is widely conserved across differentP. brassicaepathotypes, the degradation of RD21A by PbE3-2 might be a prevalent infection strategy in this pathogen.

Fusarium pseudograminearum is a pathogenic fungus causeing crown rot. Only two mycovirus have been reported in F. pseudograminearum thus far. Here, we report a fungal virus (mycovirus), Fusarium pseudograminearum fusarivirus 1 (FpFV1), isolated from the F. pseudograminearum strain ZZ-1. FpFV1 has a single-stranded positive-sense RNA (+ ssRNA) genome of 6622 nucleotides containing four open reading frames (ORFs). ORF1 encodes for a large 1,550 amino acids (aa) polypeptide with a conserved RNA-dependent RNA polymerase (RdRp) and two helicase domains. The ORF2, ORF3 and ORF4 have overlapping regions and encode putative proteins of 154 aa, 53aa and 429aa, respectively. All three ORFs have unknown functions. Based on the genomic structure, homology searches, and phylogenetic analysis, FpFV1 could be a new member of the proposed “Fusariviridae”. This is the first study to report a fusarivirus that infects F. pseudograminearum.

Chromatin transitions are mediated in part by acetylation/deacetylation post-translational modifications of histones. Histone deacetylases, e.g. sirtuins (Sir-proteins), repress transcription via promotion of heterochromatin formation. Here, we characterize the Sir2 class III histone deacetylase (BbSir2) in the environmentally and economically important fungal insect pathogen, Beauveria bassiana. BbSir2 is shown to contribute to the deacetylation of lysine residues on H3 and H4 histones. Targeted gene knockout of BbSir2 resulted in impaired asexual development, reduced abilities to utilize various carbon/nitrogen sources, reduced tolerance to oxidative, heat, and UV stress, and attenuated virulence. ΔBbSir2 cells showed disrupted cell cycle development and abnormal hyphal septation patterns. Proteomic protein acetylation analyses of wild type and ΔBbSir2 cells, revealed the differential abundance of 462 proteins, and altered (hyper- or hypo-) acetylation of 436 lysine residues on 350 proteins. Bioinformatic analyses revealed enrichment in pathways involved in carbon/nitrogen metabolism, cell cycle control and cell rescue, defense, and mitochondrial functioning. Critical targets involved in virulence included LysM effector proteins and a benzoquinone oxidoreductase implicated in detoxification of cuticular compounds. These data indicate broad effects of BbSir2 on fungal development and stress response, with identification of discrete targets that can account for the observed (decreased) virulence phenotype. This article is protected by copyright. All rights reserved.

Sclerotinia sclerotiorum ourmiavirus 17 (SsOV17) was isolated from the hypovirulent strain GF3 of Sclerotinia sclerotiorum. The genome of SsOV17 is 2,802 nt in length and contains a single long open reading frame (ORF) flanked by a short structured 5'-untranslated region (5'-UTR) (28 nt) and a long 3'-UTR (788 nt), respectively. The ORF encodes a protein with 663 amino acids and a predicted molecular mass of 75.0 kDa. A BLASTp search indicated that the protein encoded by SsOV17 is closely related to the putative RNA-dependent RNA polymerase (RdRp) of Sclerotinia sclerotiorum ourmiavirus 13 (71% identity). A multiple sequence alignment indicated that eight conserved amino acid motifs were present in the RdRp conserved region of SsOV17. Phylogenetic analysis demonstrated that SsOV17 clustered with members of the genus Botoulivirus.

Sclerotinia sclerotiorum and Botrytis cinerea are typical necrotrophic pathogens that can attack more than 700 and 3000 plant species, respectively, and cause huge economic losses across numerous crops. In particular, the absence of resistant cultivars makes the stem rot because of S. sclerotiorum the major threat of rapeseed (Brassica napus) worldwide along with Botrytis. Previously, we identified an effector‐like protein (SsSSVP1) from S. sclerotiorum and a homologue of SsSSVP1 on B. cinerea genome and found that SsSSVP1 could interact with BnQCR8 of rapeseed, a subunit of the cytochrome b‐c1 complex. In this study, we found that BnQCR8 has eight homologous copies in rapeseed cultivar Westar and reduced the copy number of BnQCR8 using CRISPR/Cas9 to improve rapeseed resistance against S. sclerotiorum. Mutants with one or more copies of BnQCR8 edited showed strong resistance against S. sclerotiorum and B. cinerea. BnQCR8‐edited mutants did not show significant difference from Westar in terms of respiration and agronomic traits tested, including the plant shape, flowering time, silique size, seed number, thousand seed weight and seed oil content. These traits make it possible to use these mutants directly for commercial production. Our study highlights a common gene for breeding of rapeseed to unravel the key hindrance of rapeseed production caused by S. sclerotiorum and B. cinerea. In contrast to previously established methodologies, our findings provide a novel strategy to develop crops with high resistance against multiple pathogens by editing only a single gene that encodes the common target of pathogen effectors.

Fusarium graminearum is an important worldwide pathogen that causes Fusarium head blight in wheat, barley, maize and other grains. LncRNAs play important roles in many biological processes, but little is known about their functions and mechanisms in filamentous fungi. Here, we report that a natural antisense RNA, GzmetE-AS, is transcribed from the opposite strand of GzmetE. GzmetE encodes a homoserine O-acetyltransferase, which is important for sexual development and plant infection. The expression of GzmetE-AS was increased significantly during the conidiation stage, while GzmetE was upregulated in the late stage of sexual reproduction. Overexpression of GzmetE-AS inhibited the transcription of GzmetE. In contrast, the expression of GzmetE was significantly increased in GzmetE-AS transcription termination strain GzmetE-AS-T. Furthermore, GzmetE-AS-T produced more perithecia and facilitated the ascospore discharge, resembling the phenotype of GzmetE overexpressing strains. However, overexpression of GzmetE-AS in ∆dcl1/2 strain cannot inhibit the expression of GzmetE, and the GzmetE nat-siRNA is also significantly reduced in ∆dcl1/2 mutant. Taken together, we have identified a novel antisense lncRNA GzmetE-AS, which is involved in asexual and sexual reproduction by regulating its antisense gene GzmetE through RNAi pathway. Our findings reveal that the lncRNA plays critical roles in the development of F. graminearum.

Clubroot, a soil-infective disease caused by

Rhabdoviruses are ubiquitous and diverse viruses that propagate owing to bidirectional interactions with their vertebrate, arthropod, and plant hosts, and some of them could pose global health or agricultural threats. However, rhabdoviruses have rarely been reported in fungi. Here, two newly identified fungal rhabdoviruses, Rhizoctonia solani rhabdovirus 1 (RsRhV1) and RsRhV2, were discovered and molecularly characterized from the phytopathogenic fungus Rhizoctonia solani. The genomic organizations of RsRhV1 and RsRhV2 are 11,716 and 11,496 nucleotides (nt) in length, respectively, and consist of five open reading frames (ORFs) (ORFs I to V). ORF I, ORF IV, and ORF V encode the viral nucleocapsid (N), glycoprotein (G), and RNA polymerase (L), respectively. The putative protein encoded by ORF III has a lower level of identity with the matrix protein of rhabdoviruses. ORF II encodes a hypothetical protein with unknown function. Phylogenetic trees based on multiple alignments of N, L, and G proteins revealed that RsRhV1 and RsRhV2 are new members of the family Rhabdoviridae, but they form an independent evolutionary branch significantly distinct from other known nonfungal rhabdoviruses, suggesting that they represent a novel viral evolutionary lineage within Rhabdoviridae. Compared to strains lacking rhabdoviruses, strains harboring RsRhV2 and RsRhV1 showed hypervirulence, suggesting that RsRhV1 and RsRhV2 might be associated with the virulence of R. solani. Taken together, this study enriches our understanding of the diversity and host range of rhabdoviruses. IMPORTANCE Mycoviruses have been attracting an increasing amount of attention due to their impact on important medical, agricultural, and industrial fungi. Rhabdoviruses are prevalent across a wide spectrum of hosts, from plants to invertebrates and vertebrates. This study molecularly characterized two novel rhabdoviruses from four Rhizoctonia solani strains, based on their genomic structures, transcription strategy, phylogenetic relationships, and biological impact on their host. Our study makes a significant contribution to the literature because it not only enriches the mycovirus database but also expands the known host range of rhabdoviruses. It also offers insight into the evolutionary linkage between animal viruses and mycoviruses and the transmission of viruses from one host to another. Our study will also help expand the contemporary knowledge of the classification of rhabdoviruses, as well as providing a new model to study rhabdovirus-host interactions, which will benefit the agriculture and medical areas of human welfare.

RNA viruses usually have linear genomes and are encapsidated by their own capsids. Here, we newly identified four mycoviruses and two previously reported mycoviruses (a fungal reovirus and a botybirnavirus) in the hypovirulent strain SCH941 of Sclerotinia sclerotiorum. One of the newly discovered mycoviruses, Sclerotinia sclerotiorum yadokarivirus 1 (SsYkV1), with a nonsegmented positive-sense single-stranded RNA (+ssRNA) genome, was molecularly characterized. SsYkV1 is 5,256 nucleotides (nt) in length, excluding the poly(A) structure, and has a large open reading frame that putatively encodes a polyprotein with the RNA-dependent RNA polymerase (RdRp) domain and a 2A-like motif. SsYkV1 was phylogenetically positioned into the family Yadokariviridae and was most closely related to Rosellinia necatrix yadokarivirus 2 (RnYkV2), with 40.55% identity (78% coverage). Although SsYkV1 does not encode its own capsid protein, the RNA and RdRp of SsYkV1 are trans-encapsidated in virions of Sclerotinia sclerotiorum botybirnavirus 3 (SsBV3), a bisegmented double-stranded RNA (dsRNA) mycovirus within the genus Botybirnavirus. In this way, SsYkV1 likely replicates inside the heterocapsid comprised of the SsBV3 capsid protein, like a dsRNA virus. SsYkV1 has a limited impact on the biological features of S. sclerotiorum. This study represents an example of a yadokarivirus trans-encapsidated by an unrelated dsRNA virus, which greatly deepens our knowledge and understanding of the unique life cycles of RNA viruses. IMPORTANCE RNA viruses typically encase their linear genomes in their own capsids. However, a capsidless +ssRNA virus (RnYkV1) highjacks the capsid of a nonsegmented dsRNA virus for the trans-encapsidation of its own RNA and RdRp. RnYkV1 belongs to the family Yadokariviridae, which already contains more than a dozen mycoviruses. However, it is unknown whether other yadokariviruses except RnYkV1 are also hosted by a heterocapsid, although dsRNA viruses with capsid proteins were detected in fungi harboring yadokarivirus. It is noteworthy that almost all presumed partner dsRNA viruses of yadokariviruses belong to the order Ghabrivirales (most probably a totivirus or toti-like virus). Here, we found a capsidless +ssRNA mycovirus, SsYkV1, from hypovirulent strain SCH941 of S. sclerotiorum, and the RNA and RdRp of this mycovirus are trans-encapsidated in virions of a bisegmented dsRNA virus within the free-floating genus Botybirnavirus. Our results greatly expand our knowledge of the unique life cycles of RNA viruses.

Mycoviruses are viruses that infect fungi, and hypovirulence-associated mycoviruses have the potential to control fungal diseases. However, it is unclear how mycovirus-mediated hypovirulent strains live and survive in the field, and no mycovirus has been applied for field crop protection. In this study, we found that a previously identified small DNA mycovirus (SsHADV-1) can convert its host, Sclerotinia sclerotiorum, from a typical necrotrophic pathogen to a beneficial endophytic fungus. SsHADV-1 downregulates the expression of key pathogenicity factor genes in S. sclerotiorum during infection. When growing in rapeseed, the SsHADV-1-infected strain DT-8 significantly regulates the expression of rapeseed genes involved in defense, hormone signaling, and circadian rhythm pathways. As a result, plant growth is promoted and disease resistance is enhanced. Field experiments showed that spraying DT-8 at the early flowering stage can reduce the disease severity of rapeseed stem rot by 67.6% and improve yield by 14.9%. Moreover, we discovered that SsHADV-1 could also infect other S. sclerotiorum strains on DT-8-inoculated plants and that DT-8 could be recovered from dead plants. These findings suggest that the mycoviruses may have the ability to shape the origin of endophytism. Our discoveries suggest that mycoviruses may influence the origin of endophytism and may also offer a novel strategy for disease control in which mycovirus-infected strains are used to improve crop health and release mycoviruses into the field.

Fungal pathogens are seriously threatening food security and natural ecosystems; efficient and environmentally friendly control methods are essential to help safeguard such resources for increasing human populations on a global scale. Here, we find that Sclerotinia sclerotiorum, a widespread pathogen of dicotyledons, can grow endophytically in wheat, rice, barley, maize, and oat, providing protection against Fusarium head blight, stripe rust, and rice blast. Protection is also provided by disabled S. sclerotiorum strains harboring a hypovirulence virus. The disabled strain DT-8 promoted wheat yields by 4–18% in the field and consistently reduced Fusarium disease by 40–60% across multiple field trials. We term the host-dependent trophism of S. sclerotiorum, destructively pathogenic or mutualistically endophytic, as schizotrophism. As a biotroph, S. sclerotiorum modified the expression of wheat genes involved in disease resistance and photosynthesis and increased the level of IAA. Our study shows that a broad-spectrum pathogen of one group of plants may be employed as a biocontrol agent in a different group of plants where they can be utilized as beneficial microorganisms while avoiding the risk of in-field release of pathogens. Our study also raises provocative questions about the potential role of schizotrophic endophytes in natural ecosystems.

Sclerotial development is a vital stage in the life cycles of many fungal plant pathogens. In this study, the protein Ss-CAD, which contains three conserved domains of cinnamyl alcohol dehydrogenase (CAD), was found to be required for sclerotial development in Sclerotinia sclerotiorum. Ss-CAD was significantly upregulated during early stage of sclerotial development. In Ss-CAD-silenced strains, sclerotial development was abnormal. In these silenced strains, formation of sclerotia was delayed or sclerotia yield was reduced, whereas hyphal growth and virulence were normal. Nox1, Nox2, and NoxR, which encode reactive oxygen species (ROS)-generating NADPH oxidases, were downregulated in Ss-CAD-silenced strains. NoxR-silenced strains displayed similar defects during sclerotial formation as Ss-CAD-silenced strains. Treatment of Ss-CAD-silenced strains with exogenous oxidants or NADPH restored normal sclerotial development. Sclerogenesis in Ss-CAD-silenced strains could also be recovered through Nox1 overexpression. The results suggest that Ss-CAD is linked to the NADPH oxidase pathways to affect sclerotial development in S. sclerotiorum.

The symbiosis of endophytes and plants is universal in nature. However, how endophytes grow in plants is not entirely clear. Previously, we reported that a virus-infected fungal pathogen could grow in plants as an endophyte. In this study, we utilized Sclerotinia sclerotiorum strain DT-8, a virus-mediated endophyte, to investigate the mechanism of symbiosis with rapeseed by dual unique molecular identifier-RNA sequencing (dual-UMI RNA-seq). We found that the expressions of genes encoding S. sclerotiorum amylase/glucoamylase, glucose transporters, and rapeseed s ugars w ill e ventually be e xported t ransporter 11 (SWEET11) were upregulated. It suggested that strain DT-8 might utilize plant starch as a nutrient. The defense systems of rapeseed were also activated, such as production of reactive oxygen species, phenylpropanoids, and brassinin, to control the growth of strain DT-8, while strain DT-8 counteracted host suppression by producing effector-like proteins, detoxification enzymes, and antioxidant components. Moreover, rapeseed also upregulated pectate lyase and pectinesterase genes to facilitate the colonization by strain DT-8. Our findings provide novel insights into the interaction of virus-mediated endophytes and their hosts that warrant further study. IMPORTANCE Although endophytes are widespread in nature, the interactions between endophytes and their hosts are still not fully understood. Members of a unique class of endophytes, the virus-mediated endophytic fungi, are continuously being discovered and have received wide attention. In this study, we investigated the interaction between a mycovirus-mediated endophytic fungus and its host rapeseed by using dual-UMI RNA-seq. According to the dual-UMI RNA-seq results, an aerial view of symbiotic mechanism under balanced regulation was suggested. This research expands our understanding of the symbiotic mechanisms of virus-fungus-plant interactions and could establish a foundation for the further development of practical application with virus-mediated hypovirulent fungi.

A novel positive single-stranded RNA virus, Sclerotinia sclerotiorum hypovirus 9 (SsHV9), was identified in the plant-pathogenic Sclerotinia sclerotiorum strain GB375, which was associated with a garden bean plant in the United States. The complete genome of SsHV9 is 14,067 nucleotides in length, excluding the poly(A) tail. It has a single large open reading frame encoding a putative polyprotein (4,196 amino acids), which is predicted to contain a papain-like protease, a protein of unknown function, an RNA-dependent RNA polymerase, and an RNA helicase. Phylogenetic analysis based on a multiple alignment of amino acid sequences of polyproteins that suggested SsHV9 belongs to the proposed genus "Alphahypovirus" in the family Hypoviridae.

Mycoviruses are an important component of the virosphere, but our current knowledge of their genome organization diversity and evolution remains rudimentary. In this study, the mycovirus composition in a hypovirulent strain of Sclerotinia sclerotiorum was molecularly characterized. Nine mycoviruses were identified and assigned into eight potential families. Of them, six were close relatives of known mycoviruses, while the other three had unique genome organizations and evolutionary positions. A deltaflexivirus with a tripartite genome has evolved via arrangement and horizontal gene transfer events, which could be an evolutionary connection from unsegmented to segmented RNA viruses. Two mycoviruses had acquired a second helicase gene by two different evolutionary mechanisms. A rhabdovirus representing an independent viral evolutionary branch was the first to be confirmed to occur naturally in fungi. The major hypovirulence-associated factor, an endornavirus, was finally corroborated. Our study expands the diversity of mycoviruses and potential virocontrol agents, and also provides new insights into virus evolutionary modes including virus genome segmentation., Author summary Identification of mycoviruses in phytopathogenic fungi is necessary for understanding the origin of viruses and developing virocontrol strategies to protect plants. Nine mycoviruses with RNA genomes were identified in a hypovirulent strain of Sclerotinia sclerotiorum and were classified into eight potential viral families, suggesting that the composition of mycoviral communities was complex in this single fungal strain. They included four previously characterized mycoviruses and three distant relatives of known mycoviruses, as well as the first reports of a deltaflexivirus with a tripartite genome, and a fungal rhabdovirus. In addition, we found an endornavirus associated with hypovirulence in a phytopathogenic fungus. Our study makes a significant contribution because it not only expands the diversity-related knowledge of mycoviruses and potential virocontrol agents, but also provides new insights into mycovirus evolution.

The infection by a single-stranded DNA virus, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), causes hypovirulence, a reduced growth rate, and other colony morphological changes in its host Sclerotinia sclerotiorum strain DT-8. However, the mechanisms of the decline are still unclear. Using digital RNA sequencing, a transcriptome analysis was conducted to elucidate the phenotype-related genes with expression changes in response to SsHADV-1 infection. A total of 3110 S. sclerotiorum differentially expressed genes (DEGs) were detected during SsHADV-1 infection, 1741 of which were up-regulated, and 1369 were down-regulated. The identified DEGs were involved in several important pathways. DNA replication, DNA damage response, carbohydrate and lipid metabolism, ribosomal assembly, and translation were the affected categories in S. sclerotiorum upon SsHADV-1 infection. Moreover, the infection of SsHADV-1 also suppressed the expression of antiviral RNA silencing and virulence factor genes. These results provide further detailed insights into the effects of SsHADV-1 infection on the whole genome transcription in S. sclerotiorum.

The infection by a single-stranded DNA virus, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), causes hypovirulence, a reduced growth rate, and other colony morphological changes in its host Sclerotinia sclerotiorum strain DT-8. However, the mechanisms of the decline are still unclear. Using digital RNA sequencing, a transcriptome analysis was conducted to elucidate the phenotype-related genes with expression changes in response to SsHADV-1 infection. A total of 3110 S. sclerotiorum differentially expressed genes (DEGs) were detected during SsHADV-1 infection, 1741 of which were up-regulated, and 1369 were down-regulated. The identified DEGs were involved in several important pathways. DNA replication, DNA damage response, carbohydrate and lipid metabolism, ribosomal assembly, and translation were the affected categories in S. sclerotiorum upon SsHADV-1 infection. Moreover, the infection of SsHADV-1 also suppressed the expression of antiviral RNA silencing and virulence factor genes. These results provide further detailed insights into the effects of SsHADV-1 infection on the whole genome transcription in S. sclerotiorum.

Rice sheath blight caused by Rhizoctonia solani is the major disease of rice that seriously threatens food security worldwide. Efficient and eco-friendly biological approaches are urgently needed since no resistant cultivars are available. In this study, fallow and paddy soils were initially subjected to microbiome analyses, and the results showed that Talaromyces spp. were significantly more abundant in the paddy soil, while Trichoderma spp. were more abundant in the fallow soil, suggesting that Talaromyces spp. could live and survive better in the paddy soil. Five isolates, namely TF-04, TF-03, TF-02, TF-01, and TA-02, were isolated from the paddy soil using sclerotia of R. solani as baits and were further evaluated for their activity against rice sheath blight. These isolates efficiently parasitized the hyphae and rotted the sclerotia even at higher water contents in sterilized sand and the soil. Isolate TF-04 significantly promoted rice growth, reduced the severity of rice sheath blight, and increased rice yield under outdoor conditions. Defense related genes were upregulated and enzyme activities were enhanced in rice treated with isolate TF-04. Our research supplies a microbiome guided approach to screen biological control agents, provides Talaromyces isolates to biologically control rice sheath blight. This article is protected by copyright. All rights reserved.

Plasmodiophora brassicae is an important biotrophic eukaryotic plant pathogen and a member of the rhizarian protists. This biotrophic pathogen causes clubroot in cruciferous plants via novel intracellular mechanisms that are markedly different from those of other biotrophic organisms. To date, genomes from six single spore isolates of P. brassicae have been sequenced. An accurate description of the evolutionary status of this biotrophic protist, however, remains lacking. Here, we determined the draft genome of the P. brassicae ZJ-1 strain. A total of 10,951 protein-coding genes were identified from a 24.1 Mb genome sequence. We applied a comparative genomics approach to prove the Rhizaria supergroup is an independent branch in the eukaryotic evolutionary tree. We also found that the GPCR signaling pathway, the versatile signal transduction to multiple intracellular signaling cascades in response to extracellular signals in eukaryotes, is significantly enriched in P. brassicae-expanded and P. brassicae-specific gene sets. Additionally, treatment with a GPCR inhibitor relieved the symptoms of clubroot and significantly suppressed the development of plasmodia. Our findings suggest that GPCR signal transduction pathways play important roles in the growth, development, and pathogenicity of P. brassicae.

Mycovirus infection is a universal phenomenon in the major fungus groups. So far, however, mycoviruses have not been described in the phytopathogenic fungus Leptosphaeria biglobosa, the causal agent of phoma stem canker (blackleg). Here, we report the complete genome sequence of a novel mitovirus, Leptosphaeria biglobosa mitovirus 1 (LbMV1), isolated from the strain H3-38 of L. biglobosa. The LbMV1 genome comprises 2568 nucleotides with a low G+C content of 30%. Using the mitochondrial genetic code, the genome of LbMV1 was found to contain a single large open reading frame that encodes a predicted protein of 756 amino acids. Multiple alignment of the predicted protein sequence showed the highest similarity (51% identity) to the putative RNA-dependent RNA polymerase of Ophiostoma mitovirus 4. In addition, phylogenetic analysis grouped LbMV1 within the genus Mitovirus of the family Narnaviridae. Our results suggest that the double-stranded RNA found in strain H3-38 is the replicating genome of the mitovirus LbMV1, its deletion represents the first evidence of a mycovirus infecting L. biglobosa.