RICEBLAST-NETWORKS · Post-transcriptional networks regulating organ-specific and general infection mechanisms in the rice blast fungus

Post-transcriptional networks regulating organ-specific and general infection mechanisms in the rice blast fungus

Blast disease caused by the ascomycetous fungus Magnaporthe oryzae is considered the most serious disease of rice worldwide. This fungal species also infects wheat, finger millet and maize. Research in Dr. Ane Sesma lab is focused in the molecular mechanisms underpinning Magnaporthe oryzae-rice interaction. M. oryzae can infect both leaf and root tissues, and represents an excellent pathosystem for studying the distinct organ-specific mechanisms involved in plant colonisation. Using the resources previously generated in Sesma lab, a multidisciplinary approach that includes transcriptomics, bioinformatics, cell biology and biochemistry aims to expand our knowledge on post-transcriptional networks that regulate organ-specific and general infection processes in the rice blast fungus. We have carried out a comparative analysis of changes in global gene expression using infected rice leaves and roots with a wild-type M. oryzae strain. We are now looking at the expression of early induced genes during in planta growth to identify genes implicated in the adjustment of the fungus to the plant environment. This has allow us to identify global changes in the fungal and plant transcriptome due to the recognition/response of different plant organs and the effect of time in gene expression. In yeast, several transcription factors are exported to the cytoplasm by Msn5p (M. oryzae EXP5 orthologue). Maturation and export of ncRNAs is also mediated by EXP5 orthologues in all organisms studied to date. We have identified proteins and RNAs that directly interact with M. oryzae Exp5, immunoprecipitation experiments using EXP5-tagged with HA-FLAG. The link between EXP5 and RNA metabolism has been confirmed by northern blots using nuclear and cytoplasmic subcellular fractions of the wild type and Δexp5 and by sequencing small RNAs in both strains. To gain an insight into the mechanisms for polyadenylation and poly(A) site selection in M. oryzae, we have used a new genome-wide sequencing approach, termed “3’ T-fill” to carry out a comprehensive map of polyadenylation sites in M. oryzae. This genome-wide comparative analysis has helped us to identify i) the nucleotide context surrounding poly(A) sites in fungal pre-mRNAs; ii) the potential motif recognised by Rbp35/CfI25 and Hrp1; iii) the involvement of Rbp35/CfI25 in alternative polyadenylation; iv) Rbp35-dependent and Hrp1-dependent mRNAs, and v) a dual function of the Rbp35/CfI25 complex in the 3’ end processing of pre-mRNAs in M. oryzae. Currently, we are testing the involvement of alternative polyadenylation in M. oryzae pathogenicity with two infection-related mRNAs. In addition to APA, other roles have been assigned to metazoan CFI68 (functional orthologue of RBP35) such as splicing and mRNA export. We also are initiating studies on additional processes in which Rbp35 participates. Proteomic analysis indicates that Rbp35 interacts not only with polyadenylation factors but with other RNA-binding proteins implicated in a range of RNA-dependent processes. At present there is a lack of knowledge of the Exp5-dependent cargoes in filamentous fungi and very little is known about the post-transcriptional mechanisms that regulate fungal plant infection. The functional characterisation of Exp5, Rbp35 and the genome-wide poly(A) mapping is expanding our knowledge on novel regulatory mechanisms regulating M. oryzae plant infection. Different 3’UTR lengths from the same gene can be generated by alternative polyadenylation (APA), a mechanism that regulates the presence of cis elements in the mRNA. The cis elements present in the 3’UTRs such as miRNA target sites modulate gene expression by affecting cytoplasmic polyadenylation, subcellular localization, stability, translation and/or decay of the mRNA. Use of APA signals often eliminates large parts of the 3’UTR, enabling escape from the stronger regulatory potential of longer 3’UTRs which may impact the protein coding capacity of the message, as well as its localization, translation efficiency, and stability. Our studies are opening new avenues of research to understand post-transcriptional regulatory mechanisms in M. oryzae. Socio-economic impact The number of people suffering from chronic hunger has raised brusquely reaching one billion worldwide with the recent increase in food prices and the global economic crisis. It is expected a global population growth to more than 9 million people by 2050, and food production will have to increase between 40-60% in order to fulfil nutritional needs worldwide. One factor that has contributed to the recent food security crisis is the decline of investment in Agricultural Research, which has been followed by a reduction of crop productivity. Several recommendations have been already proposed to try to solve food security issues for the following decades. One of them it is the urgency to invest in Agricultural Research in order to increase crop productivity and reduce yield losses. This will be achieved by introducing a sustainable intensification of arable soils thanks to the knowledge generated by biological sciences, i.e. crops resistant to stresses and diseases that produce reliable yields using renewable inputs. Blast disease is considered the most serious disease of rice worldwide. This fungal species is emerging as a very serious disease in wheat and has started to invade the Asiatic continent with severe epidemics in Bangladesh. M. oryzae also infects finger millet and maize. Joint efforts and interdisciplinary approaches are necessary to identify durable control methods of blast disease in rice fields. This projects aims to improve our understanding of the disease process, which is an essential requirement for the development of effective and durable strategies of this devastating disease.

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