Brown planthopper infestation on rice reduces plant susceptibility to Meloidogyne graminicola by reducing root sugar allocation.

Plants are simultaneously attacked by different pests that rely on sugars uptake from plants. An understanding of the role of plant sugar allocation in these multipartite interactions is limited. Here, we characterized the expression patterns of sucrose transporter genes and evaluated the impact of targeted transporter gene mutants and brown planthopper (BPH) phloem-feeding and oviposition on root sugar allocation and BPH-reduced rice susceptibility to Meloidogyne graminicola. We found that the sugar transporter genes OsSUT1 and OsSUT2 are induced at BPH oviposition sites. OsSUT2 mutants showed a higher resistance to gravid BPH than to nymph BPH, and this was correlated with callose deposition, as reflected in a different effect on M. graminicola infection. BPH phloem-feeding caused inhibition of callose deposition that was counteracted by BPH oviposition. Meanwhile, this pivotal role of sugar allocation in BPH-reduced rice susceptibility to M. graminicola was validated on rice cultivar RHT harbouring BPH resistance genes Bph3 and Bph17. In conclusion, we demonstrated that rice susceptibility to M. graminicola is regulated by BPH phloem-feeding and oviposition on rice through differences in plant sugar allocation.

The schizotrophic lifestyle of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a cosmopolitan and typical necrotrophic phytopathogenic fungus that infects hundreds of plant species. Because no cultivars highly resistant to S. sclerotiorum are available, managing Sclerotinia disease caused by S. sclerotiorum is still challenging. However, recent studies have demonstrated that S. sclerotiorum has a beneficial effect and can live mutualistically as an endophyte in graminaceous plants, protecting the plants against major fungal diseases. An in-depth understanding of the schizotrophic lifestyle of S. sclerotiorum during interactions with plants under different environmental conditions will provide new strategies for controlling fungal disease. In this review, we summarize the pathogenesis mechanisms of S. sclerotiorum during its attack of host plants as a destructive pathogen and discuss its lifestyle as a beneficial endophytic fungus.

The chromosome-scale reference genome and transcriptome analysis of Solanum torvum provides insights into resistance to root-knot nematodes.

Solanum torvum (Swartz) (2n = 24) is a wild Solanaceae plant with high economic value that is used as a rootstock in grafting for Solanaceae plants to improve the resistance to a soil-borne disease caused by root-knot nematodes (RKNs). However, the lack of a high-quality reference genome of S. torvum hinders research on the genetic basis for disease resistance and application in horticulture. Herein, we present a chromosome-level assembly of genomic sequences for S. torvum combining PacBio long reads (HiFi reads), Illumina short reads and Hi-C scaffolding technology. The assembled genome size is ~1.25 Gb with a contig N50 and scaffold N50 of 38.65 Mb and 103.02 Mb, respectively as well as a BUSCO estimate of 98%. GO enrichment and KEGG pathway analysis of the unique S. torvum genes, including NLR and ABC transporters, revealed that they were involved in disease resistance processes. RNA-seq data also confirmed that 48 NLR genes were highly expressed in roots and fibrous roots and that three homologous NLR genes (Sto0288260.1, Sto0201960.1 and Sto0265490.1) in S. torvum were significantly upregulated after RKN infection. Two ABC transporters, ABCB9 and ABCB11 were identified as the hub genes in response to RKN infection. The chromosome-scale reference genome of the S. torvum will provide insights into RKN resistance.

Discovery and Characterization of Putative Glycoprotein-Encoding Mycoviruses in the Bunyavirales.

Although segmented negative-sense RNA viruses (SNSRVs) have been frequently discovered in various fungi, most SNSRVs reported only the large segments. In this study, we investigated the diversity of the mycoviruses in the phytopathogenic fungus Fusarium asiaticum using the metatranscriptomic technique. We identified 17 fungal single-stranded RNA (ssRNA) viruses including nine viruses within Mitoviridae, one each in Narnaviridae, Botourmiaviridae, Hypoviridae, Fusariviridae, and Narliviridae, two in Mymonaviridae, and one trisegmented virus temporarily named Fusarium asiaticum mycobunyavirus 1 (FaMBV1). The FaMBV1 genome comprises three RNA segments, large (L), medium (M), and small (S) with 6,468, 2,639, and 1,420 nucleotides, respectively. These L, M, and S segments putatively encode the L protein, glycoprotein, and nucleocapsid, respectively. Phylogenetic analysis based on the L protein showed that FaMBV1 is phylogenetically clustered with Alternaria tenuissima negative-stranded RNA virus 2 (AtNSRV2) and Sclerotinia sclerotiorum negative-stranded RNA virus 5 (SsNSRV5) but distantly related to the members of the family Phenuiviridae. FaMBV1 could be vertically transmitted by asexual spores with lower efficiency (16.7%, 2/42). Comparison between FaMBV1-free and -infected fungal strains revealed that FaMBV1 has little effect on hyphal growth, pathogenicity, and conidium production, and its M segment is dispensable for viral replication and lost during subculture and asexual conidiation. The M and S segments of AtNSRV2 and SsNSRV5 were found using bioinformatics methods, indicating that the two fungal NSRVs harbor trisegmented genomes. Our results provide a new example of the existence and evolution of the segmented negative-sense RNA viruses in fungi. IMPORTANCE 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. Virome analysis of the Fusarium spp. that cause Fusarium head blight discovered a novel virus, Fusarium asiaticum mycobunyavirus 1 (FaMBV1), representing a novel lineage of the family Phenuiviridae. FaMBV1 harbors a trisegmented genome that putatively encodes RdRp, glycoproteins, and nucleocapsids. The putative glycoprotein was first described in fungal SNSRVs and shared homology with glycoprotein of animal phenuivirus but was dispensable for its replication in F. asiaticum. Two other trisegmented fungal SNSRVs that also encode glycoproteins were discovered, implying that three-segment bunyavirus infections may be common in fungi. These findings provide new insights into the ecology and evolution of SNSRVs, particularly those infecting fungi.

Morphological characterization reveals new insights into giant cell development of Meloidogyne graminicola on rice.

MAIN CONCLUSION: Three types of nematode-feeding sites (NFSs) caused by M. graminicola on rice were suggested, and the NFS polarized expansion stops before the full NFS maturation that occurs at adult female stage. Root-knot nematodes, Meloidogyne spp., secrete effectors and recruit host genes to establish their feeding sites giant cells, ensuring their nutrient acquisition. There is still a limited understanding of the mechanism underlying giant cell development. Here, the three-dimensional structures of M. graminicola-caused nematode-feeding sites (NFSs) on rice as well as changes in morphological features and cytoplasm density of the giant cells (GCs) during nematode parasitism were reconstructed and characterized by confocal microscopy and the Fiji software. Characterization of morphological features showed that three types of M. graminicola-caused NFSs, type I-III, were detected during parasitism at the second juvenile (J2), the third juvenile (J3), the fourth juvenile (J4) and adult female stages. Type I is the majority at all stages and type II develops into type I at J3 stage marked by its longitudinal growth. Meanwhile, NFSs underwent polarized expansion, where the lateral and longitudinal expansion ceased at later parasitic J2 stage and the non-feeding J4 stage, respectively. The investigation of giant cell cytoplasm density indicates that it reaches a peak at the midpoint of early parasitic J2 and adult female stages. Our data suggest the formation of three types of NFSs caused by M. graminicola on rice and the NFS polarized expansion stopping before full NFS maturation, which provides unprecedented spatio-temporal characterization of development of giant cells caused by a root-knot nematode.

Early Secretory Pathway-Associated Proteins SsEmp24 and SsErv25 Are Involved in Morphogenesis and Pathogenicity in a Filamentous Phytopathogenic Fungus.

Proper protein secretion is critical for fungal development and pathogenesis. However, the potential roles of proteins involved in the early secretory pathway are largely undescribed in filamentous fungi. p24 proteins are cargo receptors that cycle between the endoplasmic reticulum (ER) and Golgi apparatus in the early secretory pathway and recruit cargo proteins to nascent vesicles. This study characterized the function of two p24 family proteins (SsEmp24 and SsErv25) in a phytopathogenic fungus, Sclerotinia sclerotiorum. Both SsEmp24 and SsErv25 were upregulated during the early stages of S. sclerotiorum infection. ΔSsEmp24 mutant and ΔSsErv25 mutant displayed abnormal vegetative growth and sclerotium formation, were defective in infection cushion formation, and showed lower virulence on host plants. ΔSsEmp24 mutant had a more severe abnormal phenotype than ΔSsErv25 mutant, implying that SsEmp24 could play a central role in the early secretory pathway. Similar to their Saccharomyces cerevisiae counterparts, SsEmp24 interacted with SsErv25 and predominantly colocalized in the ER or nuclear envelope. The absence of SsEmp24 or SsErv25 led to defective in protein secretion in S. sclerotiorum, including the pathogenicity-related extracellular hydrolytic enzymes and effectors. It is proposed that SsEmp24 and SsErv25, components in the early secretory pathway, are involved in modulating morphogenesis and pathogenicity in S. sclerotiorum by mediating protein secretion. IMPORTANCE Understanding the reproduction and pathogenesis mechanism of phytopathogens could provide new opinions to effectively control fungal diseases. Although it has been known that effectors and extracellular hydrolytic enzymes secreted by phytopathogenic fungi play important roles in fungus-host interactions, the secretion system for the delivery of virulence factors to the host is still largely undescribed. Although the role of the early secretory pathway-associated p24 proteins in S. cerevisiae has been well characterized, the function of these proteins in filamentous fungi was scarcely known prior to this study. The present research provides evidence that p24 proteins participate in the reproduction and pathogenesis of phytopathogenic fungi through the mediation of protein secretion. This research advances our understanding of p24 proteins in filamentous phytopathogenic fungi. In addition, the candidate cargos of the two p24 proteins, SsEmp24 and SsErv25, were screened out by comparative proteomics, which could aid the identification of novel development and virulence-associated factors in phytopathogenic fungi.

Cyclophilin acts as a ribosome biogenesis factor by chaperoning the ribosomal protein (PlRPS15) in filamentous fungi.

The rapid transport of ribosomal proteins (RPs) into the nucleus and their efficient assembly into pre-ribosomal particles are prerequisites for ribosome biogenesis. Proteins that act as dedicated chaperones for RPs to maintain their stability and facilitate their assembly have not been identified in filamentous fungi. PlCYP5 is a nuclear cyclophilin in the nematophagous fungus Purpureocillium lilacinum, whose expression is up-regulated during abiotic stress and nematode egg-parasitism. Here, we found that PlCYP5 co-translationally interacted with the unassembled small ribosomal subunit protein, PlRPS15 (uS19). PlRPS15 contained an eukaryote-specific N-terminal extension that mediated the interaction with PlCYP5. PlCYP5 increased the solubility of PlRPS15 independent of its catalytic peptide-prolyl isomerase function and supported the integration of PlRPS15 into pre-ribosomes. Consistently, the phenotypes of the PlCYP5 loss-of-function mutant were similar to those of the PlRPS15 knockdown mutant (e.g. growth and ribosome biogenesis defects). PlCYP5 homologs in Arabidopsis thaliana, Homo sapiens, Schizosaccharomyces pombe, Sclerotinia sclerotiorum, Botrytis cinerea and Metarhizium anisopliae were identified. Notably, PlCYP5-PlRPS15 homologs from three filamentous fungi interacted with each other but not those from other species. In summary, our data disclosed a unique dedicated chaperone system for RPs by cyclophilin in filamentous fungi.

Analysis of Soluble Sugar Content in Minute Quantities of Rice Tissues by GC-MS.

Soluble sugars play key roles in plant growth, development, and adaption to the environment. Characterizing sugar content profiling of plant tissues promotes our understanding of the mechanisms underlying these plant processes. Several technologies have been developed to quantitate soluble sugar content in plant tissues; however, it is difficult with only minute quantities of plant tissues available. Here, we provide a detailed protocol for gas chromatography mass spectrometry (GC-MS)-based soluble sugar profiling of rice tissues that offers a good balance of sensitivity and reliability, and is considerably more sensitive and accurate than other reported methods. We summarize all the steps from sample collection and soluble sugar extraction to derivatization procedures of the soluble extracted sugars, instrumentation settings, and data analysis.

Correction: Dihydroxyacetone of wheat root exudates serves as an attractant for Heterodera avenae.

[This corrects the article DOI: 10.1371/journal.pone.0236317.].

Transcriptome Analysis of Eggplant Root in Response to Root-Knot Nematode Infection.

Eggplant (Solanum melongena L.), which belongs to the Solanaceae family, is an important vegetable crop. However, its production is severely threatened by root-knot nematodes (RKNs) in many countries. Solanum torvum, a wild relative of eggplant, is employed worldwide as rootstock for eggplant cultivation due to its resistance to soil-borne diseases such as RKNs. In this study, to identify the RKN defense mechanisms, the transcriptomic profiles of eggplant and Solanum torvum were compared. A total of 5360 differentially expressed genes (DEGs) were identified for the response to RKN infection. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that these DEGs are mainly involved in the processes of response to stimulus, protein phosphorylation, hormone signal transduction, and plant-pathogen interaction pathways. Many phytohormone-related genes and transcription factors (MYB, WRKY, and NAC) were differentially expressed at the four time points (ck, 7, 14, and 28 days post-infection). The abscisic acid signaling pathway might be involved in plant-nematode interactions. qRT-PCR validated the expression levels of some of the DEGs in eggplant. These findings demonstrate the nematode-induced expression profiles and provide some insights into the nematode resistance mechanism in eggplant.

Plasmodesmata play pivotal role in sucrose supply to Meloidogyne graminicola-caused giant cells in rice.

On infection, plant-parasitic nematodes establish feeding sites in roots from which they take up carbohydrates among other nutrients. Knowledge on how carbohydrates are supplied to the nematodes' feeding sites is limited. Here, gene expression analyses showed that RNA levels of OsSWEET11 to OsSWEET15 were extremely low in both Meloidogyne graminicola (Mg)-caused galls and noninoculated roots. All the rice sucrose transporter genes, OsSUT1 to OsSUT5, were either down-regulated in Mg-caused galls compared with noninoculated rice roots or had very low transcript abundance. OsSUT1 was the only gene up-regulated in galls, at 14 days postinoculation (dpi), after being highly down-regulated at 3 and 7 dpi. OsSUT4 was down-regulated at 3 dpi. No noticeable OsSUTs promoter activities were detected in Mg-caused galls of pOsSUT1 to -5::GUS rice lines. Loading experiments with carboxyfluorescein diacetate (CFDA) demonstrated that symplastic connections exist between phloem and Mg-caused giant cells (GCs). According to data from OsGNS5- and OsGSL2-overexpressing rice plants that had decreased and increased callose deposition, respectively, callose negatively affected Mg parasitism and sucrose supply to Mg-caused GCs. Our results suggest that plasmodesmata-mediated sucrose transport plays a pivotal role in sucrose supply from rice root phloem to Mg-caused GCs, and OsSWEET11 to -15 and OsSUTs are not major players in it, although further functional analysis is needed for OsSUT1 and OsSUT4.

Divergent RNA viruses in Macrophomina phaseolina exhibit potential as virocontrol agents.

Macrophomina phaseolina is an important necrotrophic phytopathogenic fungus and cause extensive damage in many oilseed crops. Twelve M.phaseolina isolates with diverse biological phenotypes were selected for a high-throughput sequencing-based metatranscriptomic and bioinformatics analysis to identify viruses infecting M.phaseolina. The analysis identified 40 partial or nearly complete viral genome segments, 31 of which were novel viruses. Among these viral sequences, 43% of the viral genomes were double-stranded RNA (dsRNA), 47% were positive single-stranded RNA (ssRNA+), and the remaining 10% were negative sense-stranded RNA (ssRNA-). The 40 viruses showed affinity to 13 distinct viral lineages, including Bunyavirales (four viruses), Totiviridae (three viruses), Chrysoviridae (five viruses), Partitiviridae (four viruses), Hypoviridae (one virus), Endornaviridae (two viruses), Tombusviridae (three viruses), Narnaviridae (one virus), Potyviridae (one virus), Bromoviridae (one virus), Virgaviridae (six viruses), 'Fusagraviridae' (five viruses), and Ourmiavirus (four viruses). Two viruses are closely related to two families, Potyviridae and Bromoviridae, which previously contained no mycovirus species. Moreover, nine novel viruses associated with M.phaseolina were identified in the family Totiviridae, Endornaviridae, and Partitiviridae. Coinfection with multiple viruses is prevalent in M.phaseolina, with each isolate harboring different numbers of viruses, ranging from three to eighteen. Furthermore, the effects of the viruses on the fungal host were analyzed according to the biological characteristics of each isolate. The results suggested that M.phaseolina hypovirus 2, M.phaseolina fusagravirus virus 1-5 (MpFV1-5), M.phaseolina endornavirus 1-2 (MpEV1-2), M.phaseolina ourmia-like virus 1-3 (MpOLV1-3), M.phaseolina mitovirus 4 (MpMV4), and M.phaseolina mycobunyavirus 1-4 (MpMBV1-4) were only detected in hypovirulent isolates. Those viruses associated with hypovirulence might be used as biological control agents as an environmentally friendly alternative to chemical fungicides. These findings considerably expand our understanding of mycoviruses in M.phaseolina and unvailed the presence of a huge difference among viruses in isolates from different hosts in distant geographical regions. Together, the present study provides new knowledge about viral evolution and fungus-virus coevolution.

Dihydroxyacetone of wheat root exudates serves as an attractant for Heterodera avenae.

Heterodera avenae, as an obligate endoparasite, causes severe yield loss in wheat (Triticum aestivum). Investigation on the mechanisms how H. avenae perceives wheat roots is limited. Here, the attractiveness of root exudates from eight plant genotypes to H. avenae were evaluated on agar plates. Results showed that the attraction of H. avenae to the root exudates from the non-host Brachypodium distachyon variety Bd21-3 was the highest, approximately 50 infective second-stage juveniles (J2s) per plate, followed by that from three H. avenae-susceptible wheat varieties, Zhengmai9023, Yanmai84 and Xiangmai25, as well as the resistant one of Xinyuan958, whereas the lowest attractive activity was observed in the two H. avenae-resistant wheat varieties, Xianmai20 (approximately 12 J2s/plate) and Liangxing66 (approximately 11 J2s/plate). Then Bd21-3, Zhengmai9023 and Heng4399 were selected for further assays as their different attractiveness and resistance to H. avenae, and attractants for H. avenae in their root exudates were characterized to be heat-labile and low-molecular compounds (LM) by behavioral bioassay. Based on these properties of the attractants, a principle of identifying attractants for H. avenae was set up. Then LM of six root exudates from the three plants with and without heating were separated and analyzed by HPLC-MS. Finally, dihydroxyacetone (DHA), methylprednisolone succinate, embelin and diethylpropionin in the root exudates were identified to be putative attractants for H. avenae according to the principle, and the attraction of DHA to H. avenae was validated by behavioral bioassay on agar. Our study enhances the recognition to the orientation mechanism of H. avenae towards wheat roots.

Biological Control of Tomato Gray Mold Caused by Botrytis Cinerea with the Entomopathogenic Fungus Metarhizium Anisopliae.

Gray mold disease caused by Botrytis cinerea is a devastating disease that leads to serious financial loss. In this study, the entomopathogenic fungus Metarhizium anisopliae that acts against the gray mold pathogen B. cinerea was evaluated. M. anisopliae produced a significant inhibition zone in front of the B. cinerea colony in the dual culture test. In addition, volatile organic compounds generated by M. anisopliae were shown to have an inhibitory effect on B. cinerea mycelia growth and reduced 41% of gray mold severity of postharvest tomatoes. The 10% concentration of the culture filtrate of M. anisopliae inhibited 88.62% of colony radial growth as well as 63.85% of sclerotia germination and all conidia germination of B. cinerea. Furthermore, the culture filtrate of M. anisopliae retained its inhibitory effect against the radial growth of B. cinerea even after heating for 15 min at 100 °C. Feasible mechanisms of M. anisopliae involved in the control of B. cinerea were explored, and it was demonstrated that the plasma membrane of B. cinerea conidia was damaged by the product of metabolism of M. anisopliae. In addition, after treating with culture filtrate of M. anisopliae, the B. cinerea phenotype was shown to be abnormal, and cell organelles of B. cinerea mycelia were damaged significantly. A significant control efficacy of M. anisopliae against tomato gray mold was detected on both the detached leaf assay (84.24%) as well as the whole plant (72.38%). In addition, a 78% reduction in tomato fruit mold was detected at a 10% treated concentration of M. anisopliae. These findings suggest that M. anisopliae possesses potential as a biocontrol agent against tomato gray mold in the greenhouse and during the postharvest stage.

Complete genome sequence of a novel victorivirus isolated from the sesame charcoal rot fungus Macrophomina phaseolina.

Macrophomina phaseolina is an important phytopathogenic fungus with a broad host range. Here, the complete genome sequence of a novel victorivirus, tentatively named Macrophomina phaseolina victorivirus 1 (MpV1), was identified from strain 2012-019 of M. phaseolina. The MpV1 genome is 5,128 nucleotides long with a predicted GC content of 62%. Sequence analysis indicated that two open reading frames (ORF 1 and 2) overlap at a tetranucleotide AUGA sequence. Proteins encoded by ORF1 and ORF2 showed significant sequence similarity to coat proteins and the RNA-dependent RNA polymerases, respectively, of members of the family Totiviridae. Analysis of the genomic structure of MpV1, homolog searches of the deduced amino acid sequences, and phylogenetic analysis indicated that MpV1 is a new member of the genus Victorivirus. As far as we know, this is the first report of the full-length nucleotide sequence of the genome of a novel victorivirus that infects M. phaseolina.

Discovery of Two Mycoviruses by High-Throughput Sequencing and Assembly of Mycovirus-Derived Small Silencing RNAs From a Hypovirulent Strain of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum, an important phytopathogenic fungus, harbors rich diversity of mycoviruses. Lately, more mycoviruses can be successfully and accurately discovered by deep sequencing, especially those that could not be detected by traditional double-stranded RNA (dsRNA) extraction. Previously, we reported that the hypovirulent S. sclerotiorum strain SZ-150 is coinfected by Sclerotinia sclerotiorum hypovirus 1 (SsHV1) and its related satellite RNA. Here, aside from SsHV1, we detected two other mycoviruses, Sclerotinia sclerotiorum botybirnavirus 3 (SsBV3/SZ-150) and Sclerotinia sclerotiorum mycotymovirus 1 (SsMTV1/SZ-150), coinfecting strain SZ-150, by deep sequencing and assembly of mycovirus-derived small RNAs and determined their full-length genomes. The genome of SsBV3/SZ-150 was found to be composed of two linear dsRNA segments, 6,212, and 5,880 bp in size, respectively. Each dsRNA segment of SsBV3/SZ-150 contains a large open reading frame (ORF) encoding RNA-dependent RNA polymerase (RdRp) and a hypothetical protein. The whole genome of SsBV3/SZ-150 shares more than 95% sequence identity with Botrytis porri botybirnavirus 1 (BpBV1) at the nucleotide (nt) or amino acid level. Thus, SsBV3/SZ-150 was assumed to be a strain of BpBV1. The genome of SsMTV1/SZ-150 consists of 6,391 nt excluding the poly(A) tail. SsMTV1/SZ-150 was predicted to contain a large ORF that encodes a putative replication-associated polyprotein (RP) with three conserved domains of viral RNA methyltransferase, viral RNA helicase, and RdRp. Phylogenetic analyses suggest that SsMTV1/SZ-150 is related, albeit distantly, to members of the family Tymoviridae. Analysis of the small RNAs derived from SsBV3/SZ-150 and SsMTV1/SZ-150 revealed that small-RNA lengths mainly range from 20 to 24 nt, with a peak at 22 nt, and the most abundant 5'-terminal nucleotide is uridine, suggesting that the Dicer 2 and Argonaute 1, two key components in the RNA inference pathway, may play important roles in the resistance to mycoviral infection in S. sclerotiorum. Neither SsBV3/SZ-150 nor SsMTV1/SZ-150 is a causal agent of hypovirulence in strain SZ-150.

A novel double-stranded RNA mycovirus that infects Macrophomina phaseolina.

Macrophomina phaseolina is a pathogenic fungus of the family Botryosphaeriaceae that causes stem rot or leaf blight in many economically important plants. Mycoviruses exist widely in fungi, but there are only a limited number of reports on mycovirus infection in M. phaseolina. A novel dsRNA virus, tentatively named "Macrophomina phaseolina fusagravirus 1" (MpFV1), was isolated from strain 2012-19 of M. phaseolina, and its molecular features were examined. The full-length cDNA of MpFV1 comprises 9,289 nucleotides with a predicted GC content of 48.1% and two discontinuous open reading frames (ORF 1 and 2). A-1 frameshift region with two typical factors, including a shifty heptamer (GGAAAAC) and an H-type pseudoknot, was predicted in the junction region of ORF1 and ORF2. The protein encoded by ORF1 shows significant similarity to a hypothetical protein, whereas ORF2 encodes an RNA-dependent RNA polymerase (RdRp) via a ribosomal frameshifting mechanism. Homology searches and phylogenetic analysis based on the RdRp sequence suggested that MpFV1 is a new member of the proposed family "Fusagraviridae".

Genome-Wide Identification and Characterization of the Cyclophilin Gene Family in the Nematophagous Fungus Purpureocillium lilacinum.

Purpureocillium lilacinum has been widely used as a commercial biocontrol agent for the control of plant parasitic nematodes. Whole genome analysis promotes the identification of functional genes and the exploration of their molecular mechanisms. The Cyclophilin (CYP) gene family belongs to the immunophillin superfamily, and has a conserved cyclophilin-like domain (CLD). CYPs are widely identified in prokaryotes and eukaryotes, and can be divided into single- and multi-domain proteins. In the present study, 10 CYP genes possessing the CLD, named PlCYP1-P10, were identified from the genome of P. lilacinum strain 36-1. Those 10 PlCYPs were predicted to have different cellular localizations in P. lilacinum. Phylogenetic and gene structure analysis revealed the evolutionary differentiation of CYPs between Ascomycotina and Saccharomycotina fungi, but conservation within the Ascomycotina fungi. Motif and gene structure distributions further support the result of phylogenetic analysis. Each PlCYP gene had a specific expression pattern in different development stages of P. lilacinum and its parasitism stage on eggs of Meloidogyne incognita. In addition, the 10 PlCYP genes exhibited different expression abundances in response to abiotic stresses, among which PlCYP4 was highly expressed at a high temperature (35 °C), while PlCYP6 was up-regulated under 5 mM of H2O2 stress. Furthermore, the heterologous expression of PlCYP4 and PlCYP6 in Escherichia coli enhanced the cellular tolerance against a high temperature and H2O2. In summary, our study indicates the potential functions of PlCYPs in virulence and the stress response, and also provides a frame for further analysis of the CYP gene family in Ascomycotina fungi.