Periodic Reporting for period 1 - OCNANO (Depicting the richest source of pathogen linked miscellaneous gene expression in orphan crops by Nanopore.)
Période du rapport: 2020-12-01 au 2022-11-30
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.
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.
Some of the major achievements accomplished in the study were:
1) Identified and confirmed that the Direct RNA Sequencing (DRS) Oxford Nanopore kits were found to be the most efficient in studying RNA methylation patterns and splicing choices, when compared to the Direct cDNA and PCR-cDNA kits tested.
2) Standardised optimal methods for efficient Total RNA and poly (A)+RNA isolation, library preparation and sequencing for Direct RNA Sequencing (DRS).
3) Identified significant presence of U6snRNAs in the small RNA fraction isolated using miRVana miRNA isolation kit (Ambion) using qPCR SYBR Green assays.
4) Confirmed that Arabidopsis U6 snRNA m6 A modification requires active FIO1 with RNA-Immunopurification studies.
5) High integrity total RNA was isolated from pathogen inoculated leaves of susceptible and resistant varieties of black pepper, after 24hrs. Post pathogen inoculation changes 48 and 72hpi indicated the efficiency of pathogen infection in susceptible varieties.
6) LC:MS/MS enabled the m6A/A ratio quantification studies in the poly (A)+RNA fraction isolated from different Arabidopsis accessions.
7) Crosses were done between FPA overexpression lines of Arabidopsis and (i) accessions with diverse NLRs; (ii) accessions with incompatible alleles; to study the ability of FPA to trigger and rescue hybrid necrosis.
8) Efficient protocol developed for adaptive sampling in Arabidopsis HMW genomic DNA. The sequencing run resulted in significant higher coverage for the NLR targets.
Maximum promotional activities of research findings from the MSCA work were done through bioRxiv, Division of Plant Sciences internal seminars, University’s press releases, annual SLS symposium and blogs in social networks (LinkedIn, Research Gate). The stimulating research findings on Arabidopsis U6 snRNA m6A modification studies was first communicated in bioRxiv with full open access and later published in eLife journal (doi: 10.7554/eLife.78808). It was also updated on Universities stories webpages (Twice the splice of life | University of Dundee, UK) and social networks (Twitter, LinkedIn, Research Gate). The analysis of the data generated by adaptive sampling is still being continued in the lab and once completed, all the data generated will be shared in public databases (ENA), and computational analyses via github.
1) Identified and confirmed that the Direct RNA Sequencing (DRS) Oxford Nanopore kits were found to be the most efficient in studying RNA methylation patterns and splicing choices, when compared to the Direct cDNA and PCR-cDNA kits tested.
2) Standardised optimal methods for efficient Total RNA and poly (A)+RNA isolation, library preparation and sequencing for Direct RNA Sequencing (DRS).
3) Identified significant presence of U6snRNAs in the small RNA fraction isolated using miRVana miRNA isolation kit (Ambion) using qPCR SYBR Green assays.
4) Confirmed that Arabidopsis U6 snRNA m6 A modification requires active FIO1 with RNA-Immunopurification studies.
5) High integrity total RNA was isolated from pathogen inoculated leaves of susceptible and resistant varieties of black pepper, after 24hrs. Post pathogen inoculation changes 48 and 72hpi indicated the efficiency of pathogen infection in susceptible varieties.
6) LC:MS/MS enabled the m6A/A ratio quantification studies in the poly (A)+RNA fraction isolated from different Arabidopsis accessions.
7) Crosses were done between FPA overexpression lines of Arabidopsis and (i) accessions with diverse NLRs; (ii) accessions with incompatible alleles; to study the ability of FPA to trigger and rescue hybrid necrosis.
8) Efficient protocol developed for adaptive sampling in Arabidopsis HMW genomic DNA. The sequencing run resulted in significant higher coverage for the NLR targets.
Maximum promotional activities of research findings from the MSCA work were done through bioRxiv, Division of Plant Sciences internal seminars, University’s press releases, annual SLS symposium and blogs in social networks (LinkedIn, Research Gate). The stimulating research findings on Arabidopsis U6 snRNA m6A modification studies was first communicated in bioRxiv with full open access and later published in eLife journal (doi: 10.7554/eLife.78808). It was also updated on Universities stories webpages (Twice the splice of life | University of Dundee, UK) and social networks (Twitter, LinkedIn, Research Gate). The analysis of the data generated by adaptive sampling is still being continued in the lab and once completed, all the data generated will be shared in public databases (ENA), and computational analyses via github.
The power of long read native RNA sequencing and HMW genomic DNA sequencing with adaptive sampling offer the potential to transform the annotation of plant immune response genes, especially the common components in plant immunity like NLRs. The utilisation of resistant source to unravel the mystery behind pathogen invasion and establishment of infection in susceptible plants by long read sequencing, gives better clarity on all signalling mechanisms happening inside the plant when exposed to such environmental biotic stress. The work can contribute towards rescue of economically relevant orphan crops from challenging environment, that ultimately aim for benefit in crop production. A broadened understanding on the mechanisms behind NLR transcription termination, will be an added contribution to researches on NLR diversity and evolution. Design of enhanced plant immunity can be accomplished using such information generated for further characterisation and engineering of NLRs. The day-to-day advancement in Oxford Nanopore Technologies possessing the surplus advantage of avoiding the need for fragmentation, copying or amplification of intact mRNA molecules, fastens the utilisation of these pocket-sized technology for studying transcriptomics. The MSCA work has the potential to renovate plant genome annotation and utilise the demonstrated approaches for building deeper knowledge on the interacting mechanisms between plants and pathogens.