Characterization of a novel gammapartitivirus infecting the phytopathogenic fungus Pyricularia oryzae.

In this study, we identified a novel double-strand RNA (dsRNA) mycovirus in Pyricularia oryzae, designated "Magnaporthe oryzae partitivirus 4" (MoPV4). The genome of MoPV4 consists of a dsRNA-1 segment encoding an RNA-dependent RNA polymerase (RdRP) and a dsRNA-2 segment encoding a capsid protein (CP). Phylogenetic analysis indicated that MoPV4 belongs to the genus Gammapartitivirus within family Partitiviridae. The particles of MoPV4 are isometric with a diameter of about 32.4 nm. Three-dimensional structure predictions indicated that the RdRP of MoPV4 forms a classical right-handed conformation, while the CP has a reclining-V shape.

Genome characterization of a novel narnavirus infecting the plant-pathogenic fungus Ustilaginoidea virens.

Mycoviruses are viruses that infect fungi and oomycetes. They are widespread in all major groups of plant-pathogenic fungi and oomycetes. To date, only the full genome of dsRNA mycoviruses and the contigs of positive-sense single-stranded RNA (+ssRNA) mycoviruses have been reported in Ustilaginoidea virens, which is the notorious causal agent of rice false smut (RFS). Here, we report the molecular characterization of a novel +ssRNA mycovirus, Ustilaginoidea virens narnavirus 4 (UvNV4), isolated from U. virens strain Uv418. UvNV4 has a genome of 3,131 nucleotides (nt) and possesses an open reading frame (ORF) predicted to encode an RNA-dependent RNA polymerase (RdRp) of 1,017 amino acids (aa) sequence with a molecular mass of 116.6 kDa. BLASTp analysis revealed that the RdRp showed 50.34% aa sequence identity to that of the previously described Zhangzhou Narna tick virus 1. Phylogenetic analysis indicated that UvNV4 is closely related to members of the family Narnaviridae. Taken together, these results clearly demonstrate that UvNV4 is a novel +ssRNA virus infecting U. virens.

Deficiency of ChPks and ChThr1 Inhibited DHN-Melanin Biosynthesis, Disrupted Cell Wall Integrity and Attenuated Pathogenicity in Colletotrichum higginsianum.

Colletotrichum higginsianum is a major pathogen causing anthracnose in Chinese flowering cabbage (Brassica parachinensis), posing a significant threat to the Chinese flowering cabbage industry. The conidia of C. higginsianum germinate and form melanized infection structures called appressoria, which enable penetration of the host plant's epidermal cells. However, the molecular mechanism underlying melanin biosynthesis in C. higginsianum remains poorly understood. In this study, we identified two enzymes related to DHN-melanin biosynthesis in C. higginsianum: ChPks and ChThr1. Our results demonstrate that the expression levels of genes ChPKS and ChTHR1 were significantly up-regulated during hyphal and appressorial melanization processes. Furthermore, knockout of the gene ChPKS resulted in a blocked DHN-melanin biosynthetic pathway in hyphae and appressoria, leading to increased sensitivity of the ChpksΔ mutant to cell-wall-interfering agents as well as decreased turgor pressure and pathogenicity. It should be noted that although the Chthr1Δ mutant still exhibited melanin accumulation in colonies and appressoria, its sensitivity to cell-wall-interfering agents and turgor pressure decreased compared to wild-type strains; however, complete loss of pathogenicity was not observed. In conclusion, our results indicate that DHN-melanin plays an essential role in both pathogenicity and cell wall integrity in C. higginsianum. Specifically, ChPks is crucial for DHN-melanin biosynthesis while deficiency of ChThr1 does not completely blocked melanin production.

Dual Transcriptome Analysis Reveals That ChATG8 Is Required for Fungal Development, Melanization and Pathogenicity during the Interaction between Colletotrichum higginsianum and Arabidopsis thaliana.

Anthracnose disease of cruciferous plants caused by Colletotrichum higginsianum is a serious fungal disease that affects cruciferous crops such as Chinese cabbage, Chinese flowering cabbage, broccoli, mustard plant, as well as the model plant Arabidopsis thaliana. Dual transcriptome analysis is commonly used to identify the potential mechanisms of interaction between host and pathogen. In order to identify differentially expressed genes (DEGs) in both the pathogen and host, the conidia of wild-type (ChWT) and Chatg8 mutant (Chatg8Δ) strains were inoculated onto leaves of A. thaliana, and the infected leaves of A. thaliana at 8, 22, 40, and 60 h post-inoculation (hpi) were subjected to dual RNA-seq analysis. The results showed that comparison of gene expression between the 'ChWT' and 'Chatg8Δ' samples detected 900 DEGs (306 upregulated and 594 down-regulated) at 8 hpi, 692 DEGs (283 upregulated and 409 down-regulated) at 22 hpi, 496 DEGs (220 upregulated and 276 down-regulated) at 40 hpi, and 3159 DEGs (1544 upregulated and 1615 down-regulated) at 60 hpi. GO and KEGG analyses found that the DEGs were mainly involved in fungal development, biosynthesis of secondary metabolites, plant-fungal interactions, and phytohormone signaling. The regulatory network of key genes annotated in the Pathogen-Host Interactions database (PHI-base) and Plant Resistance Genes database (PRGdb), as well as a number of key genes highly correlated with the 8, 22, 40, and 60 hpi, were identified during the infection. Among the key genes, the most significant enrichment was in the gene encoding the trihydroxynaphthalene reductase (THR1) in the melanin biosynthesis pathway. Both Chatg8Δ and Chthr1Δ strains showed varying degrees of reduction of melanin in appressoria and colonies. The pathogenicity of the Chthr1Δ strain was lost. In addition, six DEGs from C. higginsianum and six DEGs from A. thaliana were selected for real-time quantitative PCR (RT-qPCR) to confirm the RNA-seq results. The information gathered from this study enriches the resources available for research into the role of the gene ChATG8 during the infection of A. thaliana by C. higginsianum, such as potential links between melanin biosynthesis and autophagy, and the response of A. thaliana to different fungal strains, thereby providing a theoretical basis for the breeding of cruciferous green leaf vegetable cultivars with resistance to anthracnose disease.

Metatranscriptomic Analysis Reveals Rich Mycoviral Diversity in Three Major Fungal Pathogens of Rice.

In recent years, three major fungal diseases of rice, i.e., rice blast, rice false smut, and rice-sheath blight, have caused serious worldwide rice-yield reductions and are threatening global food security. Mycoviruses are ubiquitous in almost all major groups of filamentous fungi, oomycetes, and yeasts. To reveal the mycoviral diversity in three major fungal pathogens of rice, we performed a metatranscriptomic analysis of 343 strains, representing the three major fungal pathogens of rice, Pyricularia oryzae, Ustilaginoidea virens, and Rhizoctonia solani, sampled in southern China. The analysis identified 682 contigs representing the partial or complete genomes of 68 mycoviruses, with 42 described for the first time. These mycoviruses showed affinity with eight distinct lineages: Botourmiaviridae, Partitiviridae, Totiviridae, Chrysoviridae, Hypoviridae, Mitoviridae, Narnaviridae, and Polymycoviridae. More than half (36/68, 52.9%) of the viral sequences were predicted to be members of the families Narnaviridae and Botourmiaviridae. The members of the family Polymycoviridae were also identified for the first time in the three major fungal pathogens of rice. These findings are of great significance for understanding the diversity, origin, and evolution of, as well as the relationship between, genome structures and functions of mycoviruses in three major fungal pathogens of rice.

Genome Organizations and Functional Analyses of a Novel Gammapartitivirus from Rhizoctonia solani AG-1 IA Strain D122.

Here, we describe a novel double-stranded (ds) RNA mycovirus designated Rhizoctonia solani dsRNA virus 5 (RsRV5) from strain D122 of Rhizoctonia solani AG-1 IA, the causal agent of rice sheath blight. The RsRV5 genome consists of two segments of dsRNA (dsRNA-1, 1894 bp and dsRNA-2, 1755 bp), each possessing a single open reading frame (ORF). Sequence alignments and phylogenetic analyses showed that RsRV5 is a new member of the genus Gammapartitivirus in the family Partitiviridae. Transmission electron microscope (TEM) images revealed that RsRV5 has isometric viral particles with a diameter of approximately 20 nm. The mycovirus RsRV5 was successfully removed from strain D122 by using the protoplast regeneration technique, thus resulting in derivative isogenic RsRV5-cured strain D122-P being obtained. RsRV5-cured strain D122-P possessed the traits of accelerated mycelial growth rate, increased sclerotia production and enhanced pathogenicity to rice leaves compared with wild type RsRV5-infection strain D122. Transcriptome analysis showed that three genes were differentially expressed between two isogenic strains, D122 and D122-P. These findings provided new insights into the molecular mechanism of the interaction between RsRV5 and its host, D122 of R. solani AG-1 IA.

Functional validation of pathogenicity genes in rice sheath blight pathogen Rhizoctonia solani by a novel host-induced gene silencing system.

Rice sheath blight, caused by the soilborne fungus Rhizoctonia solani, causes severe yield losses worldwide. Elucidation of the pathogenic mechanism of R. solani is highly desired. However, the lack of a stable genetic transformation system has made it challenging to examine genes' functions in this fungus. Here, we present functional validation of pathogenicity genes in the rice sheath blight pathogen R. solani by a newly established tobacco rattle virus (TRV)-host-induced gene silencing (HIGS) system using the virulent R. solani AG-1 IA strain GD-118. RNA interference constructs of 33 candidate pathogenicity genes were infiltrated into Nicotiana benthamiana leaves with the TRV-HIGS system. Of these constructs, 29 resulted in a significant reduction in necrosis caused by GD-118 infection. For further validation of one of the positive genes, trehalose-6-phosphate phosphatase (Rstps2), stable rice transformants harbouring the double-stranded RNA (dsRNA) construct for Rstps2 were created. The transformants exhibited reduced gene expression of Rstps2, virulence, and trehalose accumulation in GD-118. We showed that the dsRNA for Rstps2 was taken up by GD-118 mycelia and sclerotial differentiation of GD-118 was inhibited. These findings offer gene identification opportunities for the rice sheath blight pathogen and a theoretical basis for controlling this disease by spray-induced gene silencing.

Transcriptome analysis reveals molecular mechanisms of sclerotial development in the rice sheath blight pathogen Rhizoctonia solani AG1-IA.

Rhizoctonia solani AG1-IA is a soil-borne necrotrophic pathogen that causes devastating rice sheath blight disease in rice-growing regions worldwide. Sclerotia play an important role in the life cycle of R. solani AG1-IA. In this study, RNA sequencing was used to investigate the transcriptomic dynamics of sclerotial development (SD) of R. solani AG1-IA. Gene ontology and pathway enrichment analyses using the Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to investigate the functions and pathways of differentially expressed genes (DEGs). Six cDNA libraries were generated, and more than 300 million clean reads were obtained and assembled into 15,100 unigenes. In total, 12,575 differentially expressed genes were identified and 34.62% (4353) were significantly differentially expressed with a FDR ≤ 0.01 and |log2Ratio| ≥ 1, which were enriched into eight profiles using Short Time-series Expression Miner. Furthermore, KEGG and gene ontology analyses suggest the DEGs were significantly enriched in several biological processes and pathways, including binding and catalytic functions, biosynthesis of ribosomes, and other biological functions. Further annotation of the DEGs using the Clusters of Orthologous Groups (COG) database found most DEGs were involved in amino acid transport and metabolism, as well as energy production and conversion. Furthermore, DEGs relevant to SD of R. solani AG1-IA were involved in secondary metabolite biosynthesis, melanin biosynthesis, ubiquitin processes, autophagy, and reactive oxygen species metabolism. The gene expression profiles of 10 randomly selected DEGs were validated by quantitative real-time reverse transcription PCR and were consistent with the dynamics in transcript abundance identified by RNA sequencing. The data provide a high-resolution map of gene expression during SD, a key process contributing to the pathogenicity of this devastating pathogen. In addition, this study provides a useful resource for further studies on the genomics of R. solani AG1-IA and other Rhizoctonia species.

Molecular Characterization of a Novel Endornavirus Conferring Hypovirulence in Rice Sheath Blight Fungus Rhizoctonia solani AG-1 IA Strain GD-2.

The complete sequence and genome organization of a novel Endornavirus from the hypovirulent strain GD-2 of Rhizoctonia solani AG-1 IA, the causal agent of rice sheath blight, were identified using a deep sequencing approach and it was tentatively named as Rhizoctonia solani endornavirus 1 (RsEV1). It was composed of only one segment that was 19,936 bp in length and was found to be the longest endornavirus genome that has been reported so far. The RsEV1 genome contained two open reading frames (ORFs): ORF1 and ORF2. ORF1 contained a glycosyltransferase 1 domain and a conserved RNA-dependent RNA polymerase domain, whereas ORF2 encoded a conserved hypothetical protein. Phylogenetic analysis revealed that RsEV1 was phylogenetically a new endogenous RNA virus. A horizontal transmission experiment indicated that RsEV1 could be transmitted from the host fungal strain GD-2 to a virulent strain GD-118P and resulted in hypovirulence in the derivative isogenic strain GD-118P-V1. Metabolomic analysis showed that 32 metabolites were differentially expressed between GD-118P and its isogenic hypovirulent strain GD-118P-V1. The differential metabolites were mainly classified as organic acids, amino acids, carbohydrates, and the intermediate products of energy metabolism. Pathway annotation revealed that these 32 metabolites were mainly involved in pentose and glucuronate interconversions and glyoxylate, dicarboxylate, starch, and sucrose metabolism, and so on. Taken together, our results showed that RsEV1 is a novel Endornavirus, and the infection of virulent strain GD-118P by RsEV1 caused metabolic disorders and resulted in hypovirulence. The results of this study lay a foundation for the biocontrol of rice sheath blight caused by R. solani AG1-IA.

Complete Nucleotide Sequence of a Partitivirus from Rhizoctonia solani AG-1 IA Strain C24.

The complete genome of a novel double-stranded (ds) RNA mycovirus, named as Rhizoctonia solani partitivirus 5 (RsPV5), isolated from rice sheath blight fungus R. solani AG-1 IA strain C24, was sequenced and analysed. RsPV5 consists of two segments, dsRNA-1 (1899 nucleotides) and dsRNA-2 (1787 nucleotides). DsRNA-1 has an open reading frame (ORF) 1 that potentially codes for a protein of 584 amino acid (aa) containing the conserved motifs of a RNA-dependent RNA polymerase (RdRp), and dsRNA-2 also contains a ORF 2, encoding a putative capsid protein (CP) of 513 aa. Phylogenetic analysis revealed that RsPV5 clustered together with six other viruses in an independent clade of the genus Alphapartitivirus, indicating that RsPV5 was a new member of the genus Alphapartitivirus, within the family Partitiviridae.

ROS and trehalose regulate sclerotial development in Rhizoctonia solani AG-1 IA.

Rhizoctonia solani AG-1 IA is the causal agent of rice sheath blight (RSB) and causes severe economic losses in rice-growing regions around the world. The sclerotia play an important role in the disease cycle of RSB. In this study, we report the effects of reactive oxygen species (ROS) and trehalose on the sclerotial development of R. solani AG-1 IA. Correlation was found between the level of ROS in R. solani AG-1 IA and sclerotial development. Moreover, we have shown the change of ROS-related enzymatic activities and oxidative burst occurs at the sclerotial initial stage. Six genes related to the ROS scavenging system were quantified in different sclerotial development stages by using quantitative RT-PCR technique, thereby confirming differential gene expression. Fluorescence microscopy analysis of ROS content in mycelia revealed that ROS were predominantly produced at the hyphal branches during the sclerotial initial stage. Furthermore, exogenous trehalose had a significant inhibitory effect on the activities of ROS-related enzymes and oxidative burst and led to a reduction in sclerotial dry weight. Taken together, the findings suggest that ROS has a promoting effect on the development of sclerotia, whereas trehalose serves as an inhibiting factor to sclerotial development in R. solani AG-1 IA.

Molecular identification of Neofabraea species associated with bull's-eye rot on apple using rolling-circle amplification of partial EF-1α sequence.

A rolling-circle amplification (RCA) method with padlock probes targeted on EF-1α regions was developed for rapid detection of apple bull's-eye rot pathogens, including Neofabraea malicorticis, N. perennans, N. kienholzii, and N. vagabunda (synonym: N. alba). Four padlock probes (PLP-Nm, PLP-Np, PLP-Nk, and PLP-Nv) were designed and tested against 28 samples, including 22 BER pathogen cultures, 4 closely related species, and 2 unrelated species that may cause serious apple decays. The assay successfully identified all the bull's-eye rot pathogenic fungi at the level of species, while no cross-reaction was observed in all target species and no false-positive reaction was observed with all strains used for reference. This study showed that the use of padlock probes and the combination of probe signal amplification by RCA provided an effective and sensitive method for the rapid identification of Neofabraea spp. The method could therefore be a useful tool for monitoring bull's-eye rot pathogens in port quarantine and orchard epidemiological studies.

Characterization of a novel dsRNA mycovirus isolated from strain A105 of Rhizoctonia solani AG-1 IA.

Rhizoctonia solani dsRNA virus 3 (RsRV3), a novel mycovirus, was isolated from the rice sheath blight pathogen Rhizoctonia solani AG-1 IA strain A105. The RsRV3 genome consists of two segments of dsRNA (dsRNA1, 1,890 bp and dsRNA2, 1,811 bp). DsRNA1 has a single open reading frame (ORF) with a putative conserved RNA-dependent RNA polymerase (RdRp) domain, and dsRNA2 comprises a single ORF, predicted to encode a coat protein. Purified viral particles of RsRV3 were isometric and measured approximately 20 nm in diameter by negative-stain transmission electron microscope (TEM). Phylogenetic analyses indicated that RsRV3 is highly similar to viruses taxonomically classified in the genus Alphapartitivirus, family Partitiviridae. Taken together, the integrative analyses of viral genomic organization, amino acid sequence alignments and phylogenetic analyses clearly demonstrate that the RsRV3 virus isolated from R. solani AG-1 IA strain A105 is classifiable as a new member of the genus Alphapartitivirus, family Partitiviridae.

Coevolutionary Dynamics of Rice Blast Resistance Gene Pi-ta and Magnaporthe oryzae Avirulence Gene AVR-Pita 1.

The Pi-ta gene in rice is effective in preventing infections by Magnaporthe oryzae strains that contain the corresponding avirulence gene, AVR-Pita1. Diverse haplotypes of AVR-Pita1 have been identified from isolates of M. oryzae from rice production areas in the United States and worldwide. DNA sequencing and mapping studies have revealed that AVR-Pita1 is highly unstable, while expression analysis and quantitative resistance loci mapping of the Pi-ta locus revealed complex evolutionary mechanisms of Pi-ta-mediated resistance. Among these studies, several Pi-ta transcripts were identified, most of which are probably derived from alternative splicing and exon skipping, which could produce functional resistance proteins that support a new concept of coevolution of Pi-ta and AVR-Pita1. User-friendly DNA markers for Pi-ta have been developed to support marker-assisted selection, and development of new rice varieties with the Pi-ta markers. Genome-wide association studies revealed a link between Pi-ta-mediated resistance and yield components suggesting that rice has evolved a complicated defense mechanism against the blast fungus. In this review, we detail the current understanding of Pi-ta allelic variation, its linkage with rice productivity, AVR-Pita allelic variation, and the coevolution of Pi-ta and AVR-Pita in Oryza species and M. oryzae populations, respectively. We also review the genetic and molecular basis of Pi-ta and AVR-Pita interaction, and its value in marker-assisted selection and engineering resistance.

Two distinct classes of protein related to GTB and RRM are critical in the sclerotial metamorphosis process of Rhizoctonia solani AG-1 IA.

Sheath blight of rice, caused by Rhizoctonia solani Kühn AG-1 IA [teleomorph: Thanatephorus cucumeris (Frank) Donk], is one of the major diseases of rice (Oryza sativa L.) worldwide. Sclerotia produced by R. solani AG-1 IA are crucial for their survival in adverse environments and further dissemination when environmental conditions become conducive. Differentially expressed genes during three stages of sclerotial metamorphosis of R. solani AG-1 IA were investigated by utilizing complementary DNA amplified fragment length polymorphism (cDNA-AFLP) technique. A total of 258 transcript derived fragments (TDFs) were obtained and sequenced, among which 253 TDFs were annotated with known functions through BLASTX by searching the GenBank database and 19 annotated TDFs were assigned into 19 secondary metabolic pathways through searching the Kyoto Encyclopedia of Genes and Genomes (KEGG) PATHWAY database. Moreover, the results of quantitative real-time PCR (qRT-PCR) analysis showed that the expression patterns of eight representative annotated TDFs were positively correlated with sclerotial metamorphosis. Sequence annotation of TDFs showed homology similarities to several genes encoding for proteins belonging to the glycosyltransferases B (GTB) and RNA recognition motif (RRM) superfamily and to other development-related proteins. Taken together, it is concluded that the members of the GTB and RRM superfamilies and several new genes involved in proteolytic process identified in this study might serve as the scavengers of free radicals and reactive oxygen species (ROS) and thus play an important role in the sclerotial metamorphosis process of R. solani AG-1 IA.

A novel mycovirus closely related to viruses in the genus Alphapartitivirus confers hypovirulence in the phytopathogenic fungus Rhizoctonia solani.

We report here the biological and molecular attributes of a novel dsRNA mycovirus designated Rhizoctonia solani partitivirus 2 (RsPV2) from strain GD-11 of R. solani AG-1 IA, the causal agent of rice sheath blight. The RsPV2 genome comprises two dsRNAs, each possessing a single ORF. Phylogenetic analyses indicated that this novel virus species RsPV2 showed a high sequence identity with the members of genus Alphapartitivirus in the family Partitiviridae, and formed a distinct clade distantly related to the other genera of Partitiviridae. Introduction of purified RsPV2 virus particles into protoplasts of a virus-free virulent strain GD-118 of R. solani AG-1 IA resulted in a derivative isogenic strain GD-118T with reduced mycelial growth and hypovirulence to rice leaves. Taken together, it is concluded that RsPV2 is a novel dsRNA virus belonging to Alphapartitivirus, with potential role in biological control of R. solani.

Comparison of different methods for total RNA extraction from sclerotia of Rhizoctonia solani

Background Rhizoctonia solani (teleomorph: Thanatephorus cucumeris) is one of the most important pathogens of rice (Oryza sativa L.) that causes severe yield losses in all rice-growing regions. Sclerotia, formed from the aggregation of hyphae, are important structures in the life cycles of R. solani and contain a large quantity of polysaccharides, lipids, proteins and pigments. In order to extract high-quality total RNA from the sclerotia of R. solani, five methods, including E.Z.N.A.™ Fungal RNA Kit, sodium dodecyl sulfate (SDS)-sodium borate, SDS-polyvinylpyrrolidone (PVP), guanidinium thiocyanate (GTC) and modified Trizol, were compared in this study. Results The electrophoresis results showed that it failed to extract total RNA from the sclerotia using modified Trizol method, whereas it could extract total RNA from the sclerotia using other four methods. Further experiments confirmed that the total RNA extracted using SDS-sodium borate, SDS-PVP and E.Z.N.A.™ Fungal RNA Kit methods could be used for RT-PCR of the specific amplification of GAPDH gene fragments, and that extracted using GTC method did not fulfill the requirement for above-mentioned RT-PCR experiment. Conclusion It is concluded that SDS-sodium borate and SDS-PVP methods were the better ones for the extraction of high-quality total RNA that could be used for future gene cloning and expression studies, whereas E.Z.N.A.™ Fungal RNA Kit was not taken into consideration when deal with a large quantity of samples because it is expensive and relatively low yield.

The complete genomic sequence of a novel mycovirus from Rhizoctonia solani AG-1 IA strain B275.

The complete genome of a novel mycovirus, Rhizoctonia solani dsRNA virus 1 (RsRV1) was sequenced and analyzed. It is composed of two dsRNA genome segments, 2379 bp and 1811 bp in length, which were referred to as RsRV1-1 and RsRV1-2, respectively. RsRV1-1 contains a single open reading frame (ORF1), which has a conserved RNA-dependent RNA polymerase (RdRp) domain, whereas RsRV1-2 contains a single ORF2, which might encode a multifunctional protein. The genome organization of RsRV1 is similar to that of members of the family Partitiviridae. However, phylogenetic analysis indicated that RsRV1 formed a distinct clade together with three other unclassified viruses, suggesting that RsRV1 may belong to a new family of dsRNA mycoviruses. This is the first report of the full-length nucleotide sequence of a novel dsRNA mycovirus, RsRV1, infecting R. solani AG-1 IA strain B275, the causal agent of rice sheath blight.

Wsp1 is downstream of Cin1 and regulates vesicle transport and actin cytoskeleton as an effector of Cdc42 and Rac1 in Cryptococcus neoformans.

Human Wiskott-Aldrich syndrome protein (WASP) is a scaffold linking upstream signals to the actin cytoskeleton. In response to intersectin ITSN1 and Rho GTPase Cdc42, WASP activates the Arp2/3 complex to promote actin polymerization. The human pathogen Cryptococcus neoformans contains the ITSN1 homolog Cin1 and the WASP homolog Wsp1, which share more homology with human proteins than those of other fungi. Here we demonstrate that Cin1, Cdc42/Rac1, and Wsp1 function in an effector pathway similar to that of mammalian models. In the cin1 mutant, expression of the autoactivated Wsp1-B-GBD allele partially suppressed the mutant defect in endocytosis, and expression of the constitutively active CDC42(Q61L) allele restored normal actin cytoskeleton structures. Similar phenotypic suppression can be obtained by the expression of a Cdc42-green fluorescent protein (GFP)-Wsp1 fusion protein. In addition, Rac1, which was found to exhibit a role in early endocytosis, activates Wsp1 to regulate vacuole fusion. Rac1 interacted with Wsp1 and depended on Wsp1 for its vacuolar membrane localization. Expression of the Wsp1-B-GBD allele restored vacuolar membrane fusion in the rac1 mutant. Collectively, our studies suggest novel ways in which this pathogenic fungus has adapted conserved signaling pathways to control vesicle transport and actin organization, likely benefiting survival within infected hosts.

Instability of the Magnaporthe oryzae avirulence gene AVR-Pita alters virulence.

The avirulence gene AVR-Pita of Magnaporthe oryzae determines the efficacy of the resistance gene Pi-ta in rice. The structures of the AVR-Pita alleles in 39 US isolates of M. oryzae were analyzed using polymerase chain reaction. A series of allele-specific primers were developed from the AVR-Pita gene to examine the presence of AVR-Pita. Orthologous alleles of the AVR-Pita gene were amplified from avirulent isolates. Sequence analysis of five alleles revealed three introns at identical positions in the AVR-Pita gene. All five alleles were predicted to encode metalloprotease proteins highly similar to the AVR-Pita protein. In contrast, the same regions of the AVR-Pita alleles were not amplified in the most virulent isolates, and significant variations of DNA sequence at the AVR-Pita allele were verified by Southern blot analysis. A Pot3 transposon was identified in the DNA region encoding the putative protease motif of the AVR-Pita protein from a field isolate B2 collected from a Pi-ta-containing cultivar Banks. These findings show that transposons can contribute to instability of AVR-Pita and is one molecular mechanism for defeating resistance genes in rice cultivar Banks.