Comprehensive Analysis of Autophagy-Related Genes in Rice Immunity against Magnaporthe oryzae.

Rice blast disease, caused by the fungus Magnaporthe oryzae, is a significant threat to rice production. Resistant cultivars can effectively resist the invasion of M. oryzae. Thus, the identification of disease-resistant genes is of utmost importance for improving rice production. Autophagy, a cellular process that recycles damaged components, plays a vital role in plant growth, development, senescence, stress response, and immunity. To understand the involvement of autophagy-related genes (ATGs) in rice immune response against M. oryzae, we conducted a comprehensive analysis of 37 OsATGs, including bioinformatic analysis, transcriptome analysis, disease resistance analysis, and protein interaction analysis. Bioinformatic analysis revealed that the promoter regions of 33 OsATGs contained cis-acting elements responsive to salicylic acid (SA) or jasmonic acid (JA), two key hormones involved in plant defense responses. Transcriptome data showed that 21 OsATGs were upregulated during M. oryzae infection. Loss-of-function experiments demonstrated that OsATG6c, OsATG8a, OsATG9b, and OsATG13a contribute to rice blast resistance. Additionally, through protein interaction analysis, we identified five proteins that may interact with OsATG13a and potentially contribute to plant immunity. Our study highlights the important role of autophagy in rice immunity and suggests that OsATGs may enhance resistance to rice blast fungus through the involvement of SA, JA, or immune-related proteins. These findings provide valuable insights for future efforts in improving rice production through the identification and utilization of autophagy-related genes.

Identification and Expression Analysis of Phosphatidylinositol Transfer Proteins Genes in Rice.

The family of phosphatidylinositol transfer proteins (PITPs) is able to bind specific lipids to carry out various biological functions throughout different stages of plant life. But the function of PITPs in rice plant is unclear. In this study, 30 PITPs were identified from rice genome, which showed differences in physicochemical properties, gene structure, conservation domains, and subcellular localization. The promoter region of the OsPITPs genes included at least one type of hormone response element, such as methyl jasmonate (Me JA) and salicylic acid (SA). Furthermore, the expression level of OsML-1, OsSEC14-3, OsSEC14-4, OsSEC14-15, and OsSEC14-19 genes were significantly affected by infection of rice blast fungus Magnaporthe oryzae. Based on these findings, it is possible that OsPITPs may be involved in rice innate immunity in response to M. oryzae infection through the Me JA and SA pathway.

The Wheat Head Blight Pathogen Fusarium graminearum Can Recruit Collaborating Bacteria from Soil.

In nature, fungal endophytes often have facultative endohyphal bacteria (FEB). Can a model plant pathogenic fungus have them, and does it affect their phenotype? We constructed a growth system/microcosm to allow an F. graminearum isolate to grow through natural soil and then re-isolated it on a gentamicin-containing medium, allowing endohyphal growth of bacteria while killing other bacteria. F. graminearum PH-1 labelled with a His1mCherry gene staining the fungal nuclei fluorescent red was used to confirm the re-isolation of the fungus. Most new re-isolates contained about 10 16SrRNA genes per fungal mCherry gene determined by qPCR. The F. graminearum + FEB holobiont isolates containing the bacteria were sub-cultured several times, and their bacterial contents were stable. Sequencing the bacterial 16SrRNA gene from several Fg-FEB holobiont isolates revealed endophytic bacteria known to be capable of nitrogen fixation. We tested the pathogenicity of one common Fg-FEB holobiont association, F. graminearum + Stenatrophomonas maltophilia, and found increased pathogenicity. The 16SrRNA gene load per fungal His1mCherry gene inside the wheat stayed the same as previously found in vitro. Finally, strong evidence was found for Fg-S. maltophilia symbiotic nitrogen fixation benefitting the fungus.

Characterization of two infection-induced transcription factors of Magnaporthe oryzae reveals their roles in regulating early infection and effector expression.

The initial stage of rice blast fungus, Magnaporthe oryzae, infection, before 36 h postinoculation, is a critical timespan for deploying pathogen effectors to overcome the host's defences and ultimately cause the disease. However, how this process is regulated at the transcription level remains largely unknown. This study functionally characterized two M. oryzae Early Infection-induced Transcription Factor genes (MOEITF1 and MOEITF2) and analysed their roles in this process. Target gene deletion and mutant phenotype analysis showed that the mutants Δmoeitf1 and Δmoeitf2 were only defective for infection growth but not for vegetative growth, asexual/sexual sporulation, conidial germination, and appressoria formation. Gene expression analysis of 30 putative effectors revealed that most effector genes were down-regulated in mutants, implying a potential regulation by the transcription factors. Artificial overexpression of two severely down-regulated effectors, T1REP and T2REP, in the mutants partially restored the pathogenicity of Δmoeitf1 and Δmoeitf2, respectively, indicating that these are directly regulated. Yeast one-hybrid assay and electrophoretic mobility shift assay indicated that Moeitf1 specifically bound the T1REP promoter and Moeitf2 specifically bound the T2REP promoter. Both T1REP and T2REP were predicted to be secreted during infection, and the mutants of T2REP were severely reduced in pathogenicity. Our results indicate crucial roles for the fungal-specific Moeitf1 and Moeitf2 transcription factors in regulating an essential step in M. oryzae early establishment after penetrating rice epidermal cells, highlighting these as possible targets for disease control.

Genetic Transformation and Green Fluorescent Protein Labeling in Ceratocystis paradoxa from Coconut.

Ceratocystis paradoxa, the causal agent of stem-bleeding disease of the coconut palm, causes great losses to the global coconut industry. As the mechanism of pathogenicity of C. paradoxa has not been determined, an exogenous gene marker was introduced into the fungus. In this study, pCT74-sGFP, which contains the green fluorescent protein (GFP) gene, and the hygromycin B resistance gene as a selective marker, was used as an expression vector. Several protoplast release buffers were compared to optimize protoplast preparation. The plasmid pCT74-sGFP was successfully transformed into the genome of C. paradoxa, which was verified using polymerase chain reaction and green fluorescence detection. The transformants did not exhibit any obvious differences from the wild-type isolates in terms of growth and morphological characteristics. Pathogenicity tests showed that the transformation process did not alter the virulence of the X-3314 C. paradoxa strain. This is the first report on the polyethylene glycol-mediated transformation of C. paradoxa carrying a 'reporter' gene GFP that was stably and efficiently expressed in the transformants. These findings provide a basis for future functional genomics studies of C. paradoxa and offer a novel opportunity to track the infection process of C. paradoxa.

MoIVD-Mediated Leucine Catabolism Is Required for Vegetative Growth, Conidiation and Full Virulence of the Rice Blast Fungus Magnaporthe oryzae.

Isovaleryl-CoA dehydrogenase (IVD), a member of the acyl-CoA dehydrogenase (ACAD) family, is a key enzyme catalyzing the conversion of isovaleryl-CoA to ß-methylcrotonyl-CoA in the third reaction of the leucine catabolism pathway and simultaneously transfers electrons to the electron-transferring flavoprotein (ETF) for ATP synthesis. We previously identified the ETF ortholog in rice blast fungus Magnaporthe oryzae (MoETF) and showed that MoETF was essential for fungal growth, conidiation and pathogenicity. To further investigate the biological function of electron-transferring proteins and clarify the role of leucine catabolism in growth and pathogenesis, we characterized MoIVD (M. oryzae isovaleryl-CoA dehydrogenase). MoIvd is highly conserved in fungi and its expression was highly induced by leucine. The Δmoivd mutants showed reduced growth, decreased conidiation and compromised pathogenicity, while the conidial germination and appressorial formation appeared normal. Consistent with a block in leucine degradation, the Δmoivd mutants accumulated isovaleric acid, grew more slowly, fully lacked pigmentation and completely failed to produce conidia on leucine-rich medium. These defects were largely rescued by raising the extracellular pH, suggesting that the accumulation of isovaleric acid contributes to the growth and conidiation defects. However, the reduced virulence of the mutants was probably due to their inability to overcome oxidative stress, since a large amount of ROS (reactive oxygen species) accumulated in infected host cell. In addition, MoIvd is localized to mitochondria and interacted with its electron receptor MoEtfb, the ß subunit of MoEtf. Taken together, our results suggest that MoIVD functions in leucine catabolism and is required for the vegetative growth, conidiation and full virulence of M. oryzae, providing the first evidence for IVD-mediated leucine catabolism in the development and pathogenesis of plant fungal pathogens.