AbstractPlants have evolved systems to defend against pathogens and adapted pathogens deliver effector proteins inside host cells to suppress these defences. Effectors are detected by immune receptors: the cytoplasmic nucleotide binding leucine-rich repeat (NB-LRR) proteins, leading to effector-triggered immunity (ETI). Using a NB-LRR gene enrichment and RNA-seq, previous studies identified over 700 NB-LRR genes in potato.
Dynamic changes in alternative splicing (AS) of NB-LRR genes have been shown in tobacco and Arabidopsis to be essential for the plant response to infection by, for example Pseudomonas. AS occurs in at least 60% of plant genes and is enriched in stress related genes, such as NB-LRR and receptor-like kinase genes. AS is also associated with mRNA stability through the activation of the nonsense-mediated decay (NMD) pathway; a conserved RNA surveillance mechanism. NMD targets aberrant endogenous transcripts for degradation. These transcripts contain features that allow them to be recognised by the NMD machinery: for example, premature termination codons (PTCs) and long 3’ UTRs. In plant defense against some pathogens, the transcripts of some resistance genes such as NB-LRR genes, are alternatively spliced and are turned over by NMD. The hypothesis is that infection alters the efficiency of NMD leading to an increase in the alternatively spliced PTC+-containing transcripts leading to efficient activation of the hypersensitive response and increased resistance.
I aim to understand a role for AS and NMD in the interaction between potato and Phytophthora infestans by focusing on post-transcriptional regulation and particularly NB-LRR genes. An in silico analysis of current potato genome and transcriptome databases (Phytozome and SpudDB) showed that some NB-LRR genes are potentially regulated by AS/NMD. However, the analysis of the transcript structures was severely limited by the available annotation from the databases. Very little is known about NMD in potato and to begin to identify potato genes and transcripts that show AS and NMD sensitivity, I firstly identified NMD-sensitive transcript orthologues of known NMD-targets in Arabidopsis as NMD reporters. I used these as reporters to optimise conditions for studying NMD in potato leaves using cycloheximide (CHX) treatment and to look for any effects on NMD of P. infestans infection in potato. Secondly, by developing UPF1-silenced N. benthamiana plants using virus induced gene silencing (VIGS), I explored the role of NMD in R/Avr interactions and disease development and found a delayed defence response. Finally, I exploited a recently developed high-quality Reference Transcriptome Dataset for potato (StRTD) to analyse RNA-seq data from a time-course of infection of DM potato leaves with P. infestans and CHX treated leaves to study NMD in potato. StRTD was developed using the RNA-seq datasets. Using stringent significance parameters, this analysis identified over 7,918 differentially expressed (DE) and 483 differentially alternatively spliced (DAS) genes during infection. NB-LRR genes were enriched among the DE but only a small number were significantly alternatively spliced. The analysis of AS/NMD using CHX treatment identified 2,645 DAS genes which also included some NB-LRR genes, other defense-related genes such as WRKY factor genes and genes involved in regulation of splicing and AS. In general, from this study only a few defense genes involved in the defence response between P. infestans and potato were identified as AS/NMD. In conclusion, I provide the gene regulation, alternative splicing and NMD landscape of potato challenged with P. infestans infection using transcript-level RNA-seq analysis and from a preliminary version of an improved potato transcriptome database.
|Date of Award||2019|
|Sponsors||The James Hutton Institute & BBSRC EASTBIO DTP Studentship|
|Supervisor||John Brown (Supervisor), Craig Simpson (Supervisor) & Ingo Hein (Supervisor)|
- Alternative splicing
- nonsense-mediated decay
- phytophthora infestans
- late blight
- post-transcriptional changes
- differential alternative splicing
- plant defense responses