Uncovering the structural stability of Magnaporthe oryzae effectors: a secretome-wide in silico analysis.

Rice blast, caused by the ascomycete fungus Magnaporthe oryzae, is a deadly disease and a major threat to global food security. The pathogen secretes small proteinaceous effectors, virulence factors, inside the host to manipulate and perturb the host immune system, allowing the pathogen to colonize and establish a successful infection. While the molecular functions of several effectors are characterized, very little is known about the structural stability of these effectors. We analyzed a total of 554 small secretory proteins (SSPs) from the M. oryzae secretome to decipher key features of intrinsic disorder (ID) and the structural dynamics of the selected putative effectors through thorough and systematic in silico studies. Our results suggest that out of the total SSPs, 66% were predicted as effector proteins, released either into the apoplast or cytoplasm of the host cell. Of these, 68% were found to be intrinsically disordered effector proteins (IDEPs). Among the six distinct classes of disordered effectors, we observed peculiar relationships between the localization of several effectors in the apoplast or cytoplasm and the degree of disorder. We determined the degree of structural disorder and its impact on protein foldability across all the putative small secretory effector proteins from the blast pathogen, further validated by molecular dynamics simulation studies. This study provides definite clues toward unraveling the mystery behind the importance of structural distortions in effectors and their impact on plant-pathogen interactions. The study of these dynamical segments may help identify new effectors as well.Communicated by Ramaswamy H. Sarma.

Explored secretome of M. oryzae for intrinsic disorder in effectorsClassified intrinsic disorder into six categoriesNoted varying degrees of disorder in apoplastic vs. cytoplasmic effectorsFound a correlation between intrinsic disorder and flexibilityDemonstrated flexibility patterns through molecular dynamics simulationsRevealed that intrinsic disorder influences effector interactionsIdentified an exceptional 100% disordered effector defying observed trends.

The secret password: Cell death-inducing proteins in filamentous phytopathogens - As versatile tools to develop disease-resistant crops.

Cell death-inducing proteins (CDIPs) are some of the secreted effector proteins manifested by filamentous oomycetes and fungal pathogens to invade the plant tissue and facilitate infection. Along with their involvement in different developmental processes and virulence, CDIPs play a crucial role in plant-pathogen interactions. As the name implies, CDIPs cause necrosis and trigger localised cell death in the infected host tissues by the accumulation of higher concentrations of hydrogen peroxide (H2O2), oxidative burst, accumulation of nitric oxide (NO), and electrolyte leakage. They also stimulate the biosynthesis of defense-related phytohormones such as salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), and ethylene (ET), as well as the expression of pathogenesis-related (PR) genes that are important in disease resistance. Altogether, the interactions result in the hypersensitive response (HR) in the host plant, which might confer systemic acquired resistance (SAR) in some cases against a vast array of related and unrelated pathogens. The CDIPs, due to their capability of inducing host resistance, are thus unique among the array of proteins secreted by filamentous plant pathogens. More interestingly, a few transgenic plant lines have also been developed expressing the CDIPs with added resistance. Thus, CDIPs have opened an interesting hot area of research. The present study critically reviews the current knowledge of major types of CDIPs identified across filamentous phytopathogens and their modes of action in the last couple of years. This review also highlights the recent breakthrough technologies in studying plant-pathogen interactions as well as crop improvement by enhancing disease resistance through CDIPs.

In silico characterization, molecular docking, and dynamic simulation of a novel fungal cell-death suppressing effector, MoRlpA as potential cathepsin B-like cysteine protease inhibitor during rice blast infection.

The blast fungus Magnaporthe oryzae is one of the most notorious pathogens affecting rice production worldwide. The cereal killer employs a special class of small secreted proteins called effectors to manipulate and perturb the host metabolism. In turn, the host plants trigger effector-triggered immunity (ETI) via localized cell death and hypersensitive response (HR). We have identified and characterized a novel secreted effector MoRlpA from M. oryzae by extensive in silico methods. The localization studies suggested that it is exclusively secreted in the host apoplasts. Interestingly, MoRlpA interacts with a protease, cathepsin B from rice with highest affinity. The 3D structural models of both the proteins were generated. Cathepsin B-like cysteine proteases are usually involved in programmed cell death (PCD) and autophagy in plants which lead to generation of HR upon infection. Our results suggest that MoRlpA interacts with rice cathepsin B-like cysteine protease and demolish the host counter-attack by suppressing cell death and HR during an active blast infection. This was further validated by molecular docking and molecular dynamic simulation analyses. The important residues involved in the rice-blast pathogen interactions were deciphered. Overall, this research highlights stable interactions between MoRlpA-OsCathB during rice blast pathogenesis and providing an insight into how this novel RlpA protease inhibitor-cum-effector modulates the host's apoplast to invade the host tissues and establish a successful infection. Thus, this research will help to develop potential fungicide to block the binding region of MoRlpA target so that the cryptic pathogen would be recognized by the host. HIGHLIGHTSFor the first time, a novel secreted effector protein, MoRlpA has been identified and characterised from M. oryzae in silicoMoRlpA contains a rare lipoprotein A-like DPBB domain which is often an enzymatic domain in other systemsMoRlpA as an apoplastic effector interacts with the rice protease OsCathB to suppress the cell death and hypersensitive response during rice blast infectionThe three-dimensional structures of both the MoRlpA and OsCathB proteins were predictedMoRlpA-OsCathB interactions were analysed by molecular docking and molecular dynamic simulation studiesCommunicated by Ramaswamy H. Sarma.

Comparative analysis of secondary metabolite gene clusters in different strains of Magnaporthe oryzae.

Rice blast caused by Magnaporthe oryzae continues to be a major constraint in rice production worldwide. Rice is one of the staple crops in India and rice blast causes huge economic losses. Interestingly, the Indian subcontinent is the centre for origin and diversity of rice as well as the Magnaporthe species complex. Secondary metabolites are known to play important role in pathogenesis and M. oryzae has high potential of genes involved in secondary metabolism but, unfortunately most of them remain uncharacterized. In the present study, we analysed the draft genome assemblies of M. oryzae strains isolated from different parts of India, for putative secondary metabolite key gene (SMKG) clusters encoding polyketide synthases, non-ribosomal peptide synthetases, diterpene cyclases and dimethylallyl tryptophan synthase. Based on the complete genome sequence of 70-15 strain and its previous reports of identified SMKGs, we have identified the key genes for the interrogated strains. Expression analysis of these genes amongst different strains indicates how they have evolved depending on the host and environmental conditions. To our knowledge, this study is first of its kind where the secondary metabolism genes and their role in functional adaptation were studied across several strains of M. oryzae.

WISH, a novel CFEM GPCR is indispensable for surface sensing, asexual and pathogenic differentiation in rice blast fungus.

We have selected and characterized a unique Conserved Fungal-specific Extra-cellular Membrane-spanning (CFEM) domain containing PTH11 like G-protein coupled receptor (GPCR), which is responsible for Water wettability, Infection, Surface sensing and Hyper-conidiation (WISH). The pathogenicity gene WISH is predicted to encode a novel seven transmembrane protein in the rice blast fungus, Magnaporthe oryzae, one of the deadliest pathogens of rice. We generated knockout mutants through a homologous recombination-based method to understand the function of the gene. These mutants are nonpathogenic due to a defect in sensing hydrophobic surface and appressorium differentiation. The mutant failed to undergo early events of pathogenesis, and appressorium development is diminished on inductive hydrophobic surface and was unable to penetrate susceptible rice leaves. The Δwish mutant did not develop any appressorium, suggesting that WISH protein is required for appressorium morphogenesis and is also involved in host surface recognition. We examined various aspects of pathogenesis and the results indicated involvement of WISH in preventing autolysis of vegetative hyphae, determining surface hydrophobicity and maintenance of cell-wall integrity. WISH gene from M. oryzae strain B157 complemented the Δwish mutant, indicating functional authenticity. Exogenous activation of cellular signaling failed to suppress the defects in Δwish mutants. These findings suggest that WISH GPCR senses diverse extracellular signals to play multiple roles and might have effects on PTH11 and MPG1 genes especially as an upstream effector of appressorium differentiation. It is for the first time that a typical GPCR containing seven transmembrane helices involved in the early events of plant pathogenesis of M. oryzae has been functionally characterized.