The proposed MSCA work involved the depiction of pathogen linked miscellaneous gene expression in resistant and susceptible sources of black pepper by Nanopore. Neglected or orphan crops like black pepper(Piper nigrum L.), has the potential to diversify the human diet, increase food production levels and trade so as to enable more sustainable and resilient agro- and horti-food systems. The economic value of this neglected crop is drastically affected (an annual crop loss of $ 4.5-7.5 million) by the dreadful foot rot disease caused by the oomycete fungus Phytophthora capsica. Surprisingly, the wild relative of black pepper, Piper colubrinum was totally resistant to this oomycete. This project was aimed to (a) annotate the transcriptomes of P.nigrum and P. colubrinum; (b) identify pathogen gene expression during infection using a capture array and (c) perform comparative analysis of P. nigrum and P. colubrinum gene expression during infection. The first part of the project was executed, after travelling to India. The rooted cuttings of resistant and susceptible plants were collected, pathogen was isolated and confirmed, and total RNA from pathogen induced wild (resistant) and cultivated (susceptible) varieties was extracted in sophisticated laboratory in India. Post pathogen inoculation changes (48hpi and 72hpi) confirmed the efficiency of pathogen induction studies. Further completion of subsequent objectives solely relied upon receival of isolated samples from India based on negotiation agreements.
Due to unforeseen delay in receiving the extracted RNA samples, an alternate workplan was suggested which aimed to study the premature transcription termination of a significant class of gene families -the Nucleotide binding Leucine rich repeat Receptors (NLRs) involved in the plant immune response to detect the intracellular invasion of pathogen effector proteins. There exists a balance maintenance in the fine control of NLRs- at low levels for pathogen surveillance, at high levels for pathogen invasion and at uncontrolled levels which trigger autoimmunity or hybrid necrosis. The premature transcription termination of NLRs is also affected by the RNA-binding protein FPA, which have an impact on plant immunity. To understand how natural variation in transcription termination of NLRs alter its function, FPA overexpression lines of Arabidopsis were crossed with accessions with diverse NLRs and accessions with incompatible alleles. The harvested seeds of individual crosses were screened for BASTA resistance. Enrichment of NLR targets to study the splicing, methylation patterns and poly (A) site choices employing adaptive sampling on GridION platforms in HMW genomic DNA resulted in significant higher coverage across Arabidopsis genome.
Research progressing towards understanding RNA processing of plant immune response genes by applying leading edge Oxford Nanopore Technologies, can further enhance crop productivity, food security, knowledge-based economy and society and contribute to the achievement of several UN Sustainable Development Goals. The study enables to unravel the genetic resistance or susceptible mechanisms during pathogen invasion which can eventually lead to the rescue of orphan crops like black pepper from the devastating pathogen. Targeting major plant immune response NLR genes to understand its genetic control and evolution, will establish an unknown aspect of plant immune system. Altogether the proposed work can add our capability to revolutionise crop protection.
