Glutamine is a non-essential amino acid that acts as a principal source of nitrogen and nucleic acid biosynthesis in living organisms. In Saccharomyces cerevisiae, glutamine synthetase catalyzes the synthesis of glutamine. To determine the role of glutamine synthetase in the development and pathogenicity of plant fungal pathogens, we used S. cerevisiae Gln1 amino acid sequence to identify its orthologs in Magnaporthe oryzae and named them MoGln1, MoGln2, and MoGln3. Deletion of MoGLN1 and MoGLN3 showed that they are not involved in the development and pathogenesis of M. oryzae. Conversely, ΔMogln2 was reduced in vegetative growth, experienced attenuated growth on Minimal Medium (MM), and exhibited hyphal autolysis on oatmeal and straw decoction and corn media. Exogenous l-glutamine rescued the growth of ΔMogln2 on MM. The ΔMogln2 mutant failed to produce spores and was nonpathogenic on barley leaves, as it was unable to form an appressorium-like structure from its hyphal tips. Furthermore, deletion of MoGLN2 altered the fungal cell wall integrity, with the ΔMogln2 mutant being hypersensitive to H₂O₂. MoGln1, MoGln2, and MoGln3 are located in the cytoplasm. Taken together, our results shows that MoGLN2 is important for vegetative growth, conidiation, appressorium formation, maintenance of cell wall integrity, oxidative stress tolerance and pathogenesis of M. oryzae.

Rice blast caused by Magnaporthe oryzae pathotype is the worst disease that leads to serious food insecurity globally. Understanding rice blast disease pathogenesis is therefore essential for the development of a blast disease mitigation strategy. Reverse phosphorylation mediated by phosphatases performs a vital function in the activation of diverse biological mechanisms within eukaryotic. However, little has been reported on the roles of PP2Cs in the virulence of blast fungus. In this current work, we deployed functional genomics and biochemical approaches to characterize type 2C protein phosphatase MoPtc6 in blast fungus. Deletion of MoPTC6 led to a drastic reduction in conidiophore development, conidia production, hyphal growth, and stress tolerance. Western blotting assay demonstrated that the phosphorylation level of MoOsm1 was decreased while MoMps1 was increased in the MoPtc6 deletion mutant, and comparative transcriptome assay revealed a higher number of expressed genes between mutant and wild type. Localization assay confirmed that MoPtc6 is sub-localized in the cytoplasm of mycelia, spores, and in the appressoria of M. oryzae. Furthermore, disruption of MoPTC6 impaired appressoria turgor pressure and glycogen utilization; more findings revealed attenuation of hyphal penetration and virulence upon deletion of MoPTC6. Generally, present findings suggested the role of MoPtc6 in the growth and virulence of M. oryzae.

Amidophosphoribosyl transferase catalyzes the first step of the purine nucleotide biosynthesis by converting 5-phosphoribosyl-1-pyrophosphate into 5-phosphoribosyl-1-amine. In this study, we identified and characterized the functions of MoAde4, an ortholog of yeast Ade4 in the rice blast fungus. MoAde4 is a 537-amino acid protein containing the GATase_6 and pribosyltran domains. Quantitative real-time PCR analysis showed MoADE4 transcripts were highly expressed during conidiation, early-infection, and late-infection stages of the fungus. Disruption of MoADE4 gene resulted in Δ Moade4 mutant exhibiting adenine, adenosine, and hypoxanthine auxotrophy on MM. Conidia quantification assays showed Δ Moade4 mutant was significantly reduced in sporulation. The conidia of Δ Moade4 mutant could still form appressoria but mostly failed to penetrate the rice cuticle. Pathogenicity test showed Δ Moade4 was completely nonpathogenic on rice and barley leaves which was attributed by failure of its infectious hyphae to colonize the host cells. The Δ Moade4 was defective in induction of strong host immunity and had its purine transporter genes repressed during in planta infection. Addition of exogenous adenine partially rescued conidiation and pathogenicity defects of the Δ Moade4 mutant on the barley and rice leaves. Localization assays showed that MoAde4 is located in the cytoplasm. Taken together, our results demonstrate that purine biosynthesis orchestrated by MoAde4 is required for fungal development, conidiation, more importantly, we found it to be essential for fungal pathogenicity not because of the appressorial formation, but appressorium penetration and host colonization during the plant infection of M. oryzae . Thus this findings suggests that purine biosynthesis could act as an important target for combating recalcitrant plant fungal pathogens.

Rice blast caused by Magnaporthe oryzae pathotype is the worst disease that leads to serious food insecurity globally. Understanding rice blast disease pathogenesis is therefore essential for development of blast disease mitigation strategy. Reverse phosphorylation mediated by phosphatases perform a vital function in the activation of diverse biological mechanisms within eukaryotic. However, little has been reported on the roles of PP2Cs in the virulence of blast fungus. In this current work, we deployed functional genomics and biochemical approach to characterize type 2C protein phosphatase MoPtc6 in blast fungus. Deletion of MoPTC6 led to drastic reduction in conidiophore development, conidia production, hyphal growth, and stresses tolerance. Western blotting assay demonstrated that phosphorylation level of MoOsm1 was decreased while MoMps1 was increased in the MoPtc6 deletion mutant, comparative transcriptome assay revealed higher number of expressed genes between mutant and wild type. Localization assay confirmed that MoPtc6 is sub localized in the cytoplasm of mycelia, spores, and in the appressoria of M. oryzae. Furthermore, disruption of MoPTC6 impaired appressoria turgor pressure and glycogen utilization; more findings revealed attenuation of hyphal penetration and virulence upon deletion of MoPTC6. Generally, present findings suggested the role of MoPtc6 in the growth and virulence of M. oryzae.

Rice blast, caused by the ascomycete fungus Magnaporthe oryzae, is one of the most problematic diseases for rice production, threatening global food security. Genetic resistance to some M. oryzae races can be achieved using major resistance genes that recognize their corresponding fungal avirulence genes. Weedy rice, a close relative of cultivated rice that competes with the crop, has evolved unique genetic mechanisms to resist M. oryzae infections; thus, weedy rice can serve as an excellent resource for blast control. In this study, we assessed disease scores of 183 F ₅ and F ₆ recombinant inbred lines (RILs) derived from a weedy rice × crop biparental mapping population and their parental lines, a Black Hull Awn weedy rice strain (PI 653413, RR14) and the aus-196 rice variety, using four distinct common U.S. blast races (IB33, IG1, IE1K, and IC17) under greenhouse conditions. All the parental lines were resistant to all blast races; however, RILs showed a wide degree of variation in resistance. Genotyping-by-sequencing of the RIL population and parents generated 1,498 single-nucleotide polymorphisms, which were used to construct a linkage map, and quantitative trait locus (QTL) mapping of blast resistance was performed using r/qtl. A single major blast resistance QTL on chromosome 12 was mapped to the Pi-ta/Pi39(t)/Ptr locus. Identification of Pi-ta/Pi-39(t)/Ptr as the key contributor to blast resistance in weedy rice provides insight into the evolution and adaptation of weedy rice and can aid in the development of blast-resistant rice varieties through marker-assisted selection.