Rice Blast Pathogen Magnaporthe Oryzae Research Articles

Pathogenecity and in-vitro Assessment of Some Fungicides on the Mycelial Growth of Rice Blast Pathogen (Magnaporthe oryzae B.Cauch) in Jigawa State, Nigeria

Pathogenecity and in-vitro Assessment of Some Fungicides on the Mycelial Growth of Rice Blast Pathogen (Magnaporthe oryzae B.Cauch) in Jigawa State, Nigeria

Antifungal potential of commercial silver nanoparticles against rice blast pathogen Magnaporthe oryzae

The rice blast caused by Magnaporthe oryzae is one of the major and recurrent threats to sustainable rice production in Bangladesh. To mitigate this problem, the present study was undertaken with the aim to investigate the inhibitory effects of commercially available silver nanoparticles (Ag-NPs) on the growth and development of Magnaporthe oryzae, and control of rice blast disease. In vitro experiments demonstrated that Ag-NPs significantly suppressed the radial growth of fungal mycelium, with the highest inhibition (83.54%) at a concentration of 250 ppm, while Trooper 75 WP at 400 ppm completely inhibited the fungal mycelial growth. The production of conidia and colony formation were also reduced significantly by Ag-NPs treatment. Furthermore, Ag-NPs exhibited a substantial inhibitory effect on the germination of M. oryzae conidia, with complete inhibition observed at concentrations between 200 and 250 ppm. Comparative analyses revealed that the fungicidal effect of Ag-NPs was superior to silver nitrate (AgNO3), suggesting the augmented activity of the nano properties. The sensitivity of M. oryzae to Ag-NPs was determined by calculating EC50 and EC95 values, which indicated a mean EC50 value of 131.5 ppm and a mean EC95 value of 276 ppm. Additionally, in vivo experiments showed that preventive and curative treatments with Ag-NPs significantly reduced disease incidence and severity in rice plants infected with M. oryzae, with efficacy comparable to that of the fungicide Trooper 75 WP. At higher concentrations of Ag-NPs, preventive treatment was found to be more effective than curative treatment in disease control. These findings highlighted the potential of Ag-NPs as an effective alternative for the management of rice blast. Ann. Bangladesh Agric. (2023) 27 (1): 17-30

13C Electron Nuclear Double Resonance Spectroscopy-Guided Molecular Dynamics Computations Reveal the Structure of the Enzyme-Substrate Complex of an Active, N-Linked Glycosylated Lipoxygenase.

Lipoxygenase (LOX) enzymes produce important cell-signaling mediators, yet attempts to capture and characterize LOX-substrate complexes by X-ray co-crystallography are commonly unsuccessful, requiring development of alternative structural methods. We previously reported the structure of the complex of soybean lipoxygenase, SLO, with substrate linoleic acid (LA), as visualized through the integration of 13C/1H electron nuclear double resonance (ENDOR) spectroscopy and molecular dynamics (MD) computations. However, this required substitution of the catalytic mononuclear, nonheme iron by the structurally faithful, yet inactive Mn2+ ion as a spin probe. Unlike canonical Fe-LOXs from plants and animals, LOXs from pathogenic fungi contain active mononuclear Mn2+ metallocenters. Here, we report the ground-state active-site structure of the native, fully glycosylated fungal LOX from rice blast pathogen Magnaporthe oryzae, MoLOX complexed with LA, as obtained through the 13C/1H ENDOR-guided MD approach. The catalytically important distance between the hydrogen donor, carbon-11 (C11), and the acceptor, Mn-bound oxygen, (donor-acceptor distance, DAD) for the MoLOX-LA complex derived in this fashion is 3.4 ± 0.1 Å. The difference of the MoLOX-LA DAD from that of the SLO-LA complex, 3.1 ± 0.1 Å, is functionally important, although is only 0.3 Å, despite the MoLOX complex having a Mn-C11 distance of 5.4 Å and a "carboxylate-out" substrate-binding orientation, whereas the SLO complex has a 4.9 Å Mn-C11 distance and a "carboxylate-in" substrate orientation. The results provide structural insights into reactivity differences across the LOX family, give a foundation for guiding development of MoLOX inhibitors, and highlight the robustness of the ENDOR-guided MD approach to describe LOX-substrate structures.

The ANIP1-OsWRKY62 module regulates both basal defense and Pi9-mediated immunity against Magnaporthe oryzae in rice

During effector-triggered immunity (ETI) against the devastating rice blast pathogen Magnaporthe oryzae, Pi9 functions as an intracellular resistance protein sensing the pathogen-secreted effector AvrPi9 in rice. Importantly, the underlying recognition mechanism(s) between Pi9 and AvrPi9 remains elusive. In this study, we identified a rice ubiquitin-like domain-containing protein (UDP), AVRPI9-INTERACTING PROTEIN 1 (ANIP1), which is directly targeted by AvrPi9 and also binds to Pi9 in plants. Phenotypic analysis of anip1 mutants and plants overexpressing ANIP1 revealed that ANIP1 negatively modulates rice basal defense against M. oryzae. ANIP1 undergoes 26S proteasome-mediated degradation, which can be blocked by both AvrPi9 and Pi9. Moreover, ANIP1 physically associates with the rice WRKY transcription factor OsWRKY62, which also interacts with AvrPi9 and Pi9 in plants. In the absence of Pi9, ANIP1 negatively regulates OsWRKY62 abundance, which can be promoted by AvrPi9. Accordingly, knocking out of OsWRKY62 in a non-Pi9 background decreased immunity against M. oryzae. However, we also observed that OsWRKY62 plays negative roles in defense against a compatible M. oryzae strain in Pi9-harboring rice. Pi9 binds to ANIP1 and OsWRKY62 to form a complex, which may help to keep Pi9 in an inactive state and weaken rice immunity. Furthermore, using competitive binding assays, we showed that AvrPi9 promotes Pi9 dissociation from ANIP1, which could be an important step toward ETI activation. Taken together, our results reveal an immune strategy whereby a UDP-WRKY module, targeted by a fungal effector, modulates rice immunity in distinct ways in the presence or absence of the corresponding resistance protein.

Phosphorylation and ubiquitination of OsWRKY31 are integral to OsMKK10-2-mediated defense responses in rice.

Mitogen-activated protein kinase (MPK) cascades play vital roles in plant innate immunity, growth, and development. Here, we report that the rice (Oryza sativa) transcription factor gene OsWRKY31 is a key component in a MPK signaling pathway involved in plant disease resistance in rice. We found that the activation of OsMKK10-2 enhances resistance against the rice blast pathogen Magnaporthe oryzae and suppresses growth through an increase in jasmonic acid and salicylic acid accumulation and a decrease of indole-3-acetic acid levels. Knockout of OsWRKY31 compromises the defense responses mediated by OsMKK10-2. OsMKK10-2 and OsWRKY31 physically interact, and OsWRKY31 is phosphorylated by OsMPK3, OsMPK4, and OsMPK6. Phosphomimetic OsWRKY31 has elevated DNA-binding activity and confers enhanced resistance to M. oryzae. In addition, OsWRKY31 stability is regulated by phosphorylation and ubiquitination via RING-finger E3 ubiquitin ligases interacting with WRKY 1 (OsREIW1). Taken together, our findings indicate that modification of OsWRKY31 by phosphorylation and ubiquitination functions in the OsMKK10-2-mediated defense signaling pathway.

A simplified spore-drop technique for rapid isolation of rice blast pathogen Magnaporthe oryzae from the infected rice leaf

Single spore isolation from a diseased sample is an essential step in obtaining a pure culture of a fungal pathogen. Rice blast disease caused by Magnaporthe oryzae is an inferior saprophytic competitor, and therefore, many fast-growing fungal or bacterial contaminants are predominant during its isolation. For isolation of M. oryzae, several methods are being followed; however, they are complex and often lead to contamination. In the present study, we have standardized an efficient method for rapid isolation of M. oryzae from the blast disease infected rice-leaf using single spore isolation by spore-drop technique. Following the spore-drop technique, pure culture for an isolate of M. oryzae was obtained quickly with the least contamination (4%), whereas the conventional spore-dilution and leaf-press method recorded 26.12 and 45.50% contamination, respectively. The spore-drop approach has yielded the single spore isolates in the shortest time (10 days) and can be used for regular rice blast pathogen isolation. This method can also be used for other sporulating pathogens successfully.

MoSnf5 Regulates Fungal Virulence, Growth, and Conidiation in Magnaporthe oryzae.

Snf5 (sucrose nonfermenting) is a core component of the SWI/SNF complexes and regulates diverse cellular processes in model eukaryotes. In plant pathogenic fungi, its biological function and underlying mechanisms remain unexplored. In this study, we investigated the biological roles of MoSnf5 in plant infection and fungal development in the rice blast pathogen Magnaporthe oryzae. The gene deletion mutants of MoSNF5 exhibited slower vegetative hyphal growth, severe defects in conidiogenesis, and impaired virulence and galactose utilization capacities. Domain dissection assays showed that the Snf5 domain and the N- and C-termini of MoSnf5 were all required for its full functions. Co-immunoprecipitation and yeast two-hybrid assays showed that MoSnf5 physically interacts with four proteins, including a transcription initiation factor MoTaf14. Interestingly, the ∆MoTaf14 mutants showed similar phenotypes as the ∆Mosnf5 mutants on fungal virulence and development. Moreover, assays on GFP-MoAtg8 expression and localization showed that both the ∆Mosnf5 and ∆MoTaf14 mutants were defective in autophagy. Taken together, MoSnf5 regulates fungal virulence, growth, and conidiation, possibly through regulating galactose utilization and autophagy in M. oryzae.

FoMC69 Gene in Fusarium oxysporum f. sp. radicis-lycopersici Is Essential for Pathogenicity by Involving Normal Function of Chlamydospores.

Fusarium oxysporum f. sp. radicis-lycopersici (Forl) causes crown and root rot disease in tomato, effecting severe economic losses. However, research on the pathogenicity genes and infection strategy of Forl is limited compared to that on F. oxysporum f. sp. lycopersici (Fol). In this study, we characterized FoMC69 gene in Forl as a homolog of MC69 required for pathogenicity in rice blast pathogen-Magnaporthe oryzae. Gene expression analysis revealed that FoMC69 expressionin Forl is higher than that in Folin planta. FoMC69-knockout mutant of Forl had significantly reduced root rot symptoms compared to the wild-type strain, and full pathogenicity was restored by complementation. By contrast, ΔFoMC69 mutant of Fol presented the same symptoms as the wild type, suggesting that FoMC69 of Forl, but not of Fol, was essential for full virulence in tomato plants. Morphological differences between the Forl and ΔFoMC69 in the roots were observed by fluorescent labeling using WGA-FITC. Chlamydospores of the ΔFoMC69 mutant of Forlcontinuously increased during infection and were three times higher than that of the wild type at 21 days post-inoculation. These observations suggest that FoMC69 of Forl is required for virulence to tomato plants by involving the normal development and germination of chlamydospores.

The rice blast fungus MoRgs1 functioning in cAMP signaling and pathogenicity is regulated by casein kinase MoCk2 phosphorylation and modulated by membrane protein MoEmc2.

GTP-binding protein (G-protein) and regulator of G-protein signaling (RGS) mediated signal transduction are critical in the growth and virulence of the rice blast pathogen Magnaporthe oryzae. We have previously reported that there are eight RGS and RGS-like proteins named MoRgs1 to MoRgs8 playing distinct and shared regulatory functions in M. oryzae and that MoRgs1 has a more prominent role compared to others in the fungus. To further explore the unique regulatory mechanism of MoRgs1, we screened a M. oryzae cDNA library for genes encoding MoRgs1-interacting proteins and identified MoCkb2, one of the two regulatory subunits of the casein kinase (CK) 2 MoCk2. We found that MoCkb2 and the sole catalytic subunit MoCka1 are required for the phosphorylation of MoRgs1 at the plasma membrane (PM) and late endosome (LE). We further found that an endoplasmic reticulum (ER) membrane protein complex (EMC) subunit, MoEmc2, modulates the phosphorylation of MoRgs1 by MoCk2. Interestingly, this phosphorylation is also essential for the GTPase-activating protein (GAP) function of MoRgs1. The balance among MoRgs1, MoCk2, and MoEmc2 ensures normal operation of the G-protein MoMagA-cAMP signaling required for appressorium formation and pathogenicity of the fungus. This has been the first report that an EMC subunit is directly linked to G-protein signaling through modulation of an RGS-casein kinase interaction.

Association between microsatellite markers and rice response to the rice blast pathogen Magnaporthe oryzae in selected Zambian rice genotypes

Magnaporthe oryzae, the rice blast disease pathogen is estimated to cause losses of up to 30% in rice production worldwide, yet its impact on rice production in Zambia remains unknown. Symptomatic rice leaf tissue collected from rice fields in Mongu District of Zambia was cultured on oatmeal agar medium and fungal colonies were purified and identified through leaf lesions and conidia using published keys. An M. oryzae isolate from this study was used to challenge twenty-eight selected Zambian rice genotypes to determine their responses to infection with the pathogen estimated by lesion numbers and sizes. Twelve of the genotypes (42.8%) were highly resistant (HR) with 1 to 2 lesions per cm2, 6 (21.4%) were moderately resistant with mean lesion numbers of 3 to 4 per cm2 while 10 (35.7%) were susceptible with lesion numbers above 5 per cm2 of leaf tissue. Polymerase chain reaction (PCR)-amplification of rice genomic DNA extracted from the 28 genotypes using selected microsatellite marker primers revealed a moderate genetic diversity with three of them showing significant genetic diversity. Allelic diversity among the selected genotypes was also confirmed from a calculated mean polymorphic information content of 0.8. A correlation between 8 of the microsatellite markers in some of the rice genotypes and their resistance response to M. oryzae was also established. Results show that some of the analysed rice genotypes have a significant potential for use as a genetic resource in breeding for varieties that are resistant to the rice blast pathogen for Zambian farmers.

Biosynthesis of Silver Nanoparticles Using Onion Endophytic Bacterium and Its Antifungal Activity against Rice Pathogen Magnaporthe oryzae.

Biosynthesis of silver nanoparticles (AgNPs) using endophytic bacteria is a safe alternative to the traditional chemical method. The purpose of this research is to biosynthesize AgNPs using endophytic bacterium Bacillus endophyticus strain H3 isolated from onion. The biosynthesized AgNPs with sizes from 4.17 to 26.9 nm were confirmed and characterized by various physicochemical techniques such as Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), UV-visible spectroscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) in addition to an energy dispersive spectrum (EDS) profile. The biosynthesized AgNPs at a concentration of 40 μg/mL had a strong antifungal activity against rice blast pathogen Magnaporthe oryzae with an inhibition rate of 88% in mycelial diameter. Moreover, the biosynthesized AgNPs significantly inhibited spore germination and appressorium formation of M. oryzae. Additionally, microscopic observation showed that mycelia morphology was swollen and abnormal when dealing with AgNPs. Overall, the current study revealed that AgNPs could protect rice plants against fungal infections.

Two nuclear effectors of the rice blast fungus modulate host immunity via transcriptional reprogramming

Pathogens utilize multiple types of effectors to modulate plant immunity. Although many apoplastic and cytoplasmic effectors have been reported, nuclear effectors have not been well characterized in fungal pathogens. Here, we characterize two nuclear effectors of the rice blast pathogen Magnaporthe oryzae. Both nuclear effectors are secreted via the biotrophic interfacial complex, translocated into the nuclei of initially penetrated and surrounding cells, and reprogram the expression of immunity-associated genes by binding on effector binding elements in rice. Their expression in transgenic rice causes ambivalent immunity: increased susceptibility to M. oryzae and Xanthomonas oryzae pv. oryzae, hemibiotrophic pathogens, but enhanced resistance to Cochliobolus miyabeanus, a necrotrophic pathogen. Our findings help remedy a significant knowledge deficiency in the mechanism of M. oryzae–rice interactions and underscore how effector-mediated manipulation of plant immunity by one pathogen may also affect the disease severity by other pathogens.

MoRBP9 Encoding a Ran-Binding Protein Microtubule-Organizing Center Is Required for Asexual Reproduction and Infection in the Rice Blast Pathogen Magnaporthe oryzae.

Like many fungal pathogens, the conidium and appressorium play key roles during polycyclic dissemination and infection of Magnaporthe oryzae. Ran-binding protein microtubule-organizing center (RanBPM) is a highly conserved nucleocytoplasmic protein. In animalia, RanBPM has been implicated in apoptosis, cell morphology, and transcription. However, the functional roles of RanBPM, encoded by MGG_00753 (named MoRBP9) in M. oryzae, have not been elucidated. Here, the deletion mutant ΔMorbp9 for MoRBP9 was generated via homologous recombination to investigate the functions of this gene. The ΔMorbp9 exhibited normal conidial germination and vegetative growth but dramatically reduced conidiation compared with the wild type, suggesting that MoRBP9 is involved in conidial production. ΔMorbp9 conidia failed to produce appressoria on hydrophobic surfaces, whereas ΔMorbp9 still developed aberrantly shaped appressorium-like structures at hyphal tips on the same surface, suggesting that MoRBP9 is involved in the morphology of appressorium-like structures from hyphal tips and is critical for development of appressorium from germ tubes. Taken together, our results indicated that MoRBP9 played a pleiotropic role in polycyclic dissemination and infection-related morphogenesis of M. oryzae.

The transcription factor Zt107320 affects the dimorphic switch, growth and virulence of the fungal wheat pathogen Zymoseptoria tritici.

Summary Zymoseptoria tritici is a filamentous fungus causing Septoria tritici blotch in wheat. The pathogen has a narrow host range and infections of grasses other than susceptible wheat are blocked early after stomatal penetration. During these abortive infections, the fungus shows a markedly different gene expression pattern. However, the underlying mechanisms causing differential gene expression during host and non‐host interactions are largely unknown, but likely include transcriptional regulators responsible for the onset of an infection programme in compatible hosts. MoCOD1, a member of the fungal Zn(II)2Cys6 transcription factor family, has been shown to directly affect pathogenicity in the rice blast pathogen Magnaporthe oryzae. Here, we analyse the role of the putative transcription factor Zt107320, a homologue of MoCOD1, during infection of compatible and incompatible hosts by Z. tritici. We show for the first time that Zt107320 is differentially expressed in host versus non‐host infections and that lower expression corresponds to an incompatible infection of non‐hosts. Applying reverse genetics approaches, we further show that Zt107320 regulates the dimorphic switch as well as the growth rate of Z. tritici and affects fungal cell wall composition in vitro. Moreover, ∆Zt107320 mutants showed reduced virulence during compatible infections of wheat. We conclude that Zt107320 directly influences pathogen fitness and propose that Zt107320 is involved in the regulation of growth processes and pathogenicity during infection.

Cross-reactivity of a rice NLR immune receptor to distinct effectors from the rice blast pathogen Magnaporthe oryzae provides partial disease resistance

Unconventional integrated domains in plant intracellular immune receptors of the nucleotide-binding leucine-rich repeat (NLRs) type can directly bind translocated effector proteins from pathogens and thereby initiate an immune response. The rice (Oryza sativa) immune receptor pairs Pik-1/Pik-2 and RGA5/RGA4 both use integrated heavy metal-associated (HMA) domains to bind the effectors AVR–Pik and AVR–Pia, respectively, from the rice blast fungal pathogen Magnaporthe oryzae. These effectors both belong to the MAX effector family and share a core structural fold, despite being divergent in sequence. How integrated domains in NLRs maintain specificity of effector recognition, even of structurally similar effectors, has implications for understanding plant immune receptor evolution and function. Here, using plant cell death and pathogenicity assays and protein–protein interaction analyses, we show that the rice NLR pair Pikp-1/Pikp-2 triggers an immune response leading to partial disease resistance toward the “mis-matched” effector AVR–Pia in planta and that the Pikp–HMA domain binds AVR–Pia in vitro. We observed that the HMA domain from another Pik-1 allele, Pikm, cannot bind AVR–Pia, and it does not trigger a plant response. The crystal structure of Pikp–HMA bound to AVR–Pia at 1.9 Å resolution revealed a binding interface different from those formed with AVR–Pik effectors, suggesting plasticity in integrated domain-effector interactions. The results of our work indicate that a single NLR immune receptor can bait multiple pathogen effectors via an integrated domain, insights that may enable engineering plant immune receptors with extended disease resistance profiles.

A Feed-Forward Subnetwork Emerging from Integrated TOR- and cAMP/PKA-Signaling Architecture Reinforces Magnaporthe oryzae Appressorium Morphogenesis.

Appressoria are important mediators of plant-microbe interactions. In the devastating rice blast pathogen Magnaporthe oryzae, appressorial morphogenesis from germ tube tips requires activated cAMP/PKA signaling and inactivated TOR signaling (TORoff). TORoff temporarily arrests G2 at a metabolic checkpoint during the single round of mitosis that occurs following germination. G2 arrest induces autophagy and appressorium formation concomitantly, allowing reprogression of the cell cycle to G1/G0 quiescence and a single appressorial nucleus. Inappropriate TOR activation abrogates G2 arrest and inhibits cAMP/PKA signaling downstream of cPKA. This results in multiple rounds of germ tube mitosis and the loss of autophagy and appressoria formation. How cAMP/PKA signaling connects to cell cycle progression and autophagy is not known. To address this, we interrogated TOR and cAMP/PKA pathways using signaling mutants, different surface properties, and specific cell cycle inhibitors and discovered a feed-forward subnetwork arising from TOR- and cAMP/PKA-signaling integration. This adenylate cyclase-cAMP-TOR-adenylate cyclase subnetwork reinforces cAMP/PKA-dependent appressorium formation under favorable environmental conditions. Under unfavorable conditions, the subnetwork collapses, resulting in reversible cell cycle-mediated germ tube growth regardless of external nutrient status. Collectively, this work provides new molecular insights on germ tube morphogenetic decision-making in response to static and dynamic environmental conditions.

Disruption of putative short-chain acyl-CoA dehydrogenases compromised free radical scavenging, conidiogenesis, and pathogenesis of Magnaporthe oryzae

Short-chain acyl-CoA dehydrogenase (Scad) mediated β-oxidation serves as the fastest route for generating essential energies required to support the survival of organisms under stress or starvation. In this study, we identified three putative SCAD genes in the genome of the globally destructive rice blast pathogen Magnaporthe oryzae, named as MoSCAD1, MoSCAD2, and MoSCAD3. To elucidate their function, we deployed targeted gene deletion strategy to investigate individual and the combined influence of MoSCAD genes on growth, stress tolerance, conidiation and pathogenicity of the rice blast fungus. First, localization and co-localization results obtained from this study showed that MoScad1 localizes to the endoplasmic reticulum (ER), MoScad2 localizes exclusively to the mitochondria while MoScad3 partially localizes to the mitochondria and peroxisome at all developmental stages of M. oryzae. Results obtained from this investigation showed that the deletion of MoSCAD1 and MoSCAD2 caused a minimal but significant reduction in the growth of ΔMoscad1 and ΔMoscad2 strains, while, growth characteristics exhibited by the ΔMoscad3 strain was similar to the wild-type strain. Furthermore, we observed that deletion of MoSCAD2 resulted in drastic reduction in conidiation, delayed conidia germination, triggered the development of abnormal appressorium and suppressed host penetration and colonization efficiencies of the ΔMoscad1 strain. This study provides first material evidence confirming the possible existence of ER β-oxidation pathway in M. oryzae. We also infer that mitochondria β-oxidation rather than peroxisomal and ER β-oxidation play an essential role in the vegetative growth, conidiation, appressorial morphogenesis and progression of pathogenesis in M. oryzae.

A Small GTPase RHO2 Plays an Important Role in Pre-infection Development in the Rice Blast Pathogen Magnaporthe oryzae.

The rice blast pathogen Magnaporthe oryzae is a global threat to rice production. Here we characterized RHO2 gene (MGG_02457) that belongs to the Rho GTPase family, using a deletion mutant. This mutant ΔMorho2 exhibited no defects in conidiation and germination but developed only 6% of appressoria in response to a hydrophobic surface when compared to the wild-type progenitor. This result indicates that MoRHO2 plays a role in appressorium development. Furthermore, exogenous cAMP treatment on the mutant led to appressoria that exhibited abnormal morphology on both hydrophobic and hydrophilic surfaces. These outcomes suggested the involvement of MoRHO2 in cAMP-mediated appressorium development. ΔMorho2 mutation also delayed the development of appressorium-like structures (ALS) at hyphal tips on hydrophobic surface, which were also abnormally shaped. These results suggested that MoRHO2 is involved in morphological development of appressoria and ALS from conidia and hyphae, respectively. As expected, ΔMorho2 mutant was defective in plant penetration, but was still able to cause lesions, albeit at a reduced rate on wounded plants. These results implied that MoRHO2 plays a role in M. oryzae virulence as well.

Distinct roles of the YPEL gene family in development and pathogenicity in the ascomycete fungus Magnaporthe oryzae

Members of the Yippee-like (YPEL) gene family are highly conserved in eukaryotes and are homologous to the Drosophila yippee gene. In this study, we functionally characterized two YPEL-homologous genes, MoYPEL1 and MoYPEL2, in the rice blast pathogen Magnaporthe oryzae using the deletion mutants ΔMoypel1, ΔMoypel2, and ΔΔMoypel1,2. The MoYPEL1 deletion mutant was significantly defective in conidiation and unable to undergo appressorium development; however, deletion of MoYPEL2 resulted in a significant increase in conidiation and the abnormal development of two appressoria per conidium. These data demonstrate the opposite roles of each member of the YPEL gene family during the development of M. oryzae. The double mutant was phenotypically similar to the ΔMoypel1 mutant in conidiation, but similar to the ΔMoypel2 mutant in appressorium development. Subcellular localization of the MoYPEL1 protein was dynamic during appressorium development, while the MoYPEL2 protein consistently localized within the nuclei during developmental stages. Our studies indicate that the two YPEL gene family members play distinct roles in the developmental stages of M. oryzae, furthering our understanding of disease dissemination and development in fungi.

Family-Four Aldehyde Dehydrogenases Play an Indispensable Role in the Pathogenesis of Magnaporthe oryzae.

The oxidative degradation of lipids through lipid peroxidation processes results in the generation of free fatty acid radicals. These free radicals including reactive oxygen species (ROS) serve as a substrate for generating reactive aldehydes. The accumulation of free fatty acid radicals, ROS, and reactive aldehydes in cell compartments beyond physiological threshold levels tends to exert a damaging effect on proximal membranes and distal tissues. Living organisms deploy a wide array of efficient enzymes including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and aldehyde dehydrogenases (ALDHs) for scavenging reactive molecules and intermediates produced from membrane lipid peroxidation events. Although the contributions of SOD, CAT, and POD to the pathogenesis of microbial plant pathogens are well known, the influence of ALDH genes on the morphological and infectious development of plant pathogenic microbes is not well understood. In this study, we deployed RNA interference (RNAi) techniques and successfully silenced two putative family-four aldehyde dehydrogenase genes potassium-activated aldehyde dehydrogenase (MoKDCDH) and delta-1-pyrrorine-5-carboxylate dehydrogenase (MoP5CDH) in the rice blast pathogen Magnaporthe oryzae. The results obtained from the phenotypic analysis of individual knock-down strains showed that the RNAi-mediated inactivation of MoKDCDH and MoP5CDH triggered a significant reduction in conidiogenesis and vegetative growth of ΔMokdcdh and ΔMop5cdh strains. We further observed that downregulating the expression of MoKDCDH and MoP5CDH severely compromised the pathogenesis of the rice blast fungus. Also, the disruption of MoKDCDH and MoP5CDH M. oryzae undermined membrane integrity and rendered the mutant strains highly sensitive to membrane stress inducing osmolytes. However, the MoKDCDH and MoP5CDH knock-down strains generated in this study displayed unaltered cell wall integrity and thus suggested that family-four ALDHs play a dispensable role in enforcing cell wall-directed stress tolerance in M. oryzae. From these results, we deduced that family-four ALDHs play a conserved role in fostering membrane integrity in M. oryzae possibly by scavenging reactive aldehydes, fatty acid radicals, and other alcohol derivatives. The observation that downregulating the expression activities of MoKDCDH had a lethal effect on potential mutants further emphasized the need for comprehensive and holistic evaluation of the numerous ALDHs amassed by the rice blast fungus for their possible engagement as suitable targets as antiblast agents.