Metabolite profiling reveals overexpression of the global regulator, MoLAEA leads to increased synthesis of metabolites in Magnaporthe oryzae.

AIMS: To study the altered metabolic pathways and metabolites produced in overexpression and knockdown mutants of a global regulator named MoLAEA, which was recently found to regulate the expression of the genes involved in secondary metabolism in one of the most destructive plant pathogens, Magnaporthe oryzae. METHODS AND RESULTS: Mass spectrometry-based global untargeted metabolomic profiling was used to identify altered metabolites. Metabolites were extracted from the mutant strains of MoLAEA using two extraction methods viz., aqueous and organic extraction and data acquired using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in positive and negative polarities. Levels of metabolites involved in various biological pathways such as amino acid as well as polyamine biosynthesis, fatty acid and pyrimidine metabolism showed a remarkable change in the mutant strains. Interestingly, metabolites involved in stress responses were produced in higher quantities in the overexpression strain, whereas certain overproduced metabolites were associated with distinctive phenotypic changes in the overexpression strain compared with the wild type. Further, the expression of several genes involved in the stress responses was found to have higher expression in the overexpression strain. CONCLUSIONS: The global regulator MoLAEA is involved in secondary metabolism in the plant pathogen M. oryzae such that the mutant strains showed an altered level of several metabolites involved in the biosynthesis pathways compared with the wild type. Also, metabolites involved in stress responses were overproduced in the overexpression strain and this can be seen in the higher growth in media amended with stress-inducing agents or a higher expression of genes involved in stress response in the overexpression strain compared with the wild type. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of metabolite profiling relative to the global regulation of secondary metabolism in M. oryzae, where secondary metabolism is poorly understood. It opens up avenues for more relevant investigations on the genetic regulation of several of the metabolites found in the analysis, which have not been previously characterized in M. oryzae.

Spray-Induced Silencing of Pathogenicity Gene MoDES1 via Exogenous Double-Stranded RNA Can Confer Partial Resistance Against Fungal Blast in Rice.

Over the past years, RNA interference (RNAi) has been used as a promising combat strategy against a wide range of pests and pathogens in ensuring global food security. It involves the induction of highly specific posttranscriptional regulation of target essential genes from an organism, via the application of precursor long, non-coding double-stranded RNA (dsRNA) molecules that share sequence-complementarity with the mRNAs of the targets. Fungal blast disease caused by Magnaporthe oryzae is one of the most deadly diseases of rice and wheat incurring huge losses in global crop yield. To date, the host-induced gene silencing (HIGS) and virus-induced gene silencing (VIGS) aspects of RNAi have been successfully exploited in developing resistance against M. oryzae in rice. Spray-induced gene silencing (SIGS) is a current, potential, non-transformative, and environment-friendly pest and pathogen management strategy, where naked or nanomaterial-bound dsRNA are sprayed on leaves to cause selective knockdown of pathogenicity genes. Although it relies on the ability of fungal pathogens to uptake sprayed RNA, its efficiency varies largely across phytopathogens and their genes, targeted for silencing. Here, we report a transient dsRNA supplementation system for the targeted knockdown of MoDES1, a host-defense suppressor pathogenicity gene from M. oryzae. We validate the feasibility of in vivo SIGS and post-uptake transfer of RNA signals to distal plant parts in rice-M. oryzae pathosystem through a GFP-based reporter system. A protocol for efficient silencing via direct foliar spray of naked dsRNA was optimized. As proof-of-concept, we demonstrate the phenotypic impacts of in vitro dsDES1 treatment on growth, conidiation, ROS-scavenging ability, and pathogenic potential of M. oryzae. Furthermore, our extrapolatory dsDES1 spray experiments on wounded leaves and whole rice plants indicate resultant silencing of MoDES1 that conferred significant resistance against the fungal blast disease. The evaluation of primary and secondary host defense responses provides evidence supporting the notion that spray of sequence-specific dsRNA on wounded leaf tissue can cause systemic and sustained silencing of a M. oryzae target gene. For the first time, we establish a transgene-free SIGS approach as a promising crop protection strategy against the notorious rice-blast fungus.

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.

Optimisation of a rapid and efficient transformation protocol for fungal blast-susceptible indica rice cultivars HR-12 and CO-39.

Rice is an important staple crop and fungal blast disease destroys about 10-30% of its global produce, annually. Although genetic manipulation has largely been employed in crop-improvement programmes and agricultural biotechnology, the ease of transformation of several recalcitrant indica cultivars continues to be a challenge. HR-12 and CO-39 are two indica cultivars that are commonly used in breeding programmes, but are susceptible to biotic threats like fungal blast and sheath blight disease. Here in this study, we have optimised a rapid and reproducible transformation protocol for the said cultivars, having compared both the tissue-culture and in-planta methods of transformation. Murashige & Skoog basal media supplemented with maltose and 2.5 mg l-1 2,4-D induced efficient callogenesis in HR-12, while maltose with 3 mg l-1 2,4-D gave optimum results in case of CO-39. The media containing 0.5 mg l-1 NAA, 3 mg l-1 BAP, and 1 mg l-1 kinetin yielded a maximum regeneration efficiency of 62% and 65% in HR-12 and CO-39, respectively. The studies with Agrobacterium tumefaciens, LBA4404 strain harbouring pCAMBIA1303 suggested that although these cultivars demonstrated successful gene-transfer, they failed to regenerate efficiently, post-transformation. Alternatively, our modified in-planta piercing and vacuum infiltration-based protocol resulted in 33-35% transformation efficiency in less than half the time required for tissue-culture based transformation method. As per our knowledge, it is among the highest obtained from existing piercing-based direct transformation protocols in rice, and can also be implemented in genetically manipulating other recalcitrant varieties of rice.

MoLAEA Regulates Secondary Metabolism in Magnaporthe oryzae.

Fungi are rich sources of secondary metabolites of pharmaceutical importance, such as antibiotics, antitumor agents, and immunosuppressants, as well as of harmful toxins. Secondary metabolites play important roles in the development and pathogenesis of fungi. LaeA is a global regulator of secondary metabolism and was originally reported in Aspergillus nidulans; however, its role in secondary metabolism in Magnaporthe oryzae has not yet been reported. Here, we investigated the role of a gene homologous to LAEA (loss of AflR expression) of Aspergillus spp. in Magnaporthe oryzae, named M. oryzaeLAEA (MoLAEA). Studies on MoLAEA overexpression and knockdown strains have suggested that this gene acts as a negative regulator of sporulation and melanin synthesis. However, it is not involved in the growth and pathogenesis of M. oryzae Transcriptomic data indicated that MoLAEA regulated genes involved in secondary metabolism. Interestingly, we observed (for the first time, to our knowledge) that this gene is involved in benzylpenicillin (penicillin G) synthesis in M. oryzae Overexpression of MoLAEA increased penicillin G production, whereas the silenced strain showed a complete absence of penicillin G compared to its presence in the wild type. We also observed that MoLaeA interacted with MoVeA, a velvet family protein involved in fungal development and secondary metabolism, in the nucleus. This study showed that though MoLAEA may not make any contribution in rice blast fungal pathogenesis, it regulates secondary metabolism in M. oryzae and thus can be further studied for identifying other new uncharacterized metabolites in this fungus.IMPORTANCEM. oryzae causes blast disease, the most serious disease of cultivated rice affecting global rice production. The genome of M. oryzae has been shown to have a number of genes involved in secondary metabolism, but most of them are uncharacterized. In fact, compared to studies of other filamentous fungi, hardly any work has been done on secondary metabolism in M. oryzae It is shown here (for the first time, to our knowledge) that penicillin G is being synthesized in M. oryzae and that MoLAEA is involved in this process. This is the first step in understanding the penicillin G biosynthesis pathway in M. oryzae This study also unraveled the details of how MoLaeA works by forming a nuclear complex with MoVeA in M. oryzae, thus indicating functional conservation of such a gene across filamentous fungi. All these findings open up avenues for more relevant investigations on the genetic regulation of secondary metabolism in 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.

Genomic resources of Magnaporthe oryzae (GROMO): a comprehensive and integrated database on rice blast fungus.

BACKGROUND: Magnaporthe oryzae, rice blast fungus, is the most devastating pathogen of rice. It has emerged as a model phytopathogen for the study of host-pathogen interactions. A large body of data has been generated on different aspects of biology of this fungus and on host-pathogen interactions. However, most of the data is scattered and is not available as a single resource for researchers in this field. DESCRIPTION: Genomic Resources of Magnaporthe oyzae (GROMO), is a specialized, and comprehensive database for rice blast fungus, integrating information from several resources. GROMO contains information on genomic sequence, mutants available, gene expression, localization of proteins obtained from a variety of repositories, as primary data. In addition, prediction of domains, pathways, protein-protein interactions, sumolyation sites and biochemical properties that were obtained after computational analysis of protein sequences have also been included as derived data. This database has an intuitive user interface that shall prompt the user to explore various possible information resources available on a given gene or a protein, from a single source. CONCLUSION: Currently, information on M. oryzae is available from different resources like BROAD MIT Magnaporthe database, Agrobacterium tumefaciens-mediated transformation (ATMT) M. oryzae database, Magnaporthe grisea--Oryza sativa (MGOS) and Massive Parallel Signature Sequencing (MPSS) databases. In the GROMO project, an effort has been made to integrate information from all these databases, derive some new data based on the available information analyzed by relevant programs and make more insightful predictions to better understand the biology of M. oryzae. The database is currently available at: http://gromo.msubiotech.ac.in/

Expression of the lipid transfer protein Ace-AMP1 in transgenic wheat enhances antifungal activity and defense responses.

To enhance fungal disease resistance, wheat plants (cv. Bobwhite) were engineered to constitutively express the potent antimicrobial protein Ace-AMP1 from Allium cepa, driven by a maize ubiquitin promoter along with its first intron. The bar gene was used for selection of putative transformants on medium containing phosphinothricin (PPT). Transgene inheritance, integration and stability of expression were confirmed over two generations by PCR, Southern, northern and western blot analyses, respectively. The levels of Ace-AMP1 in different transgenic lines correlated with the transcript levels of the transgene. Up to 50% increase in resistance to Blumeria graminis f. sp. tritici was detected in detached leaf assays. In ears of transgenic wheat inoculated with Neovossia indica, Ace-AMP1 intensified expression of defense-related genes. Elevated levels of salicylic acid and of transcripts of phenylalanine ammonia lyase (PAL), glucanase (PR2) and chitinase (PR3) in the transgenic plants indicated manifestation of systemic acquired resistance (SAR).