Transcriptome profiling of potato plant stems challenged with Pectobacterium carotovorum subsp. brasiliense and elucidation of the role of small RNAs in Pectobacterium survival mechanisms

Abstract

Pectobacterium carotovorum subsp. brasiliense, a necrotrophic phytopathogen belonging to the soft rot Enterobacteriaceae (SRE) family is responsible for causing tuber soft rot and blackleg diseases of stems in potato plants. In recent years, P. c. brasiliense, has emerged as a soft rot pathogen of significance, potentially threatening potato production globally. To date, P. c. brasiliense is the most aggressive soft rot phytopathogen isolated from potato in South Africa. Currently effective chemical control measures are unavailable once soft rot pathogens have established disease in potato plants and/or harvested tubers. Therefore, this study sought to determine the molecular basis of quantitative resistance in potato stems challenged with P. c. brasiliense. In addition, this thesis explores some of the regulatory mechanisms important in the adaptation of Pectobacterium species to harsh nutrient-deficient environments such as plant xylem vessels. Determining the activated defense responses in potato stems is key in deciphering potential control approaches against pectobacteria as these soft rot pathogens colonize vascular tissues during infection of plants. Currently, no transcriptome-wide studies have been applied in the P. c. brasiliense and potato stem interaction to understand inducible defense responses within potato stems. In chapter 2, by implementing a time-course RNA-seq analysis, our study revealed important signaling pathways suggested to contribute to the potato defense transcriptome against P. c. brasiliense infection. Comparison of transcriptomes between a susceptible potato cultivar (Solanum tuberosum cv Valor) and tolerant cultivar (S. tuberosum cv BP1) following P. c. brasiliense inoculation revealed that the MAPK signaling cascades and ethylene hormonal pathway are central to potato defense responses against this pathogen. Specifically, genes encoding MPK3 protein kinase, and MKS1; ethylene biosynthetic and signaling pathways such as ACC, ERF2 and EIN3 genes were up-regulated in the tolerant cultivar within the time-course. Furthermore, expression of downstream defense-related genes was enhanced in S. tuberosum cv BP1, including transcription factors such WRKY33, MYB83, and several ethylene-responsive binding factors (ERFs); as well as various secondary wall biosynthetic genes for lignification and cellulose biosynthesis, for example, IRX9 and CESA8, respectively. In chapter 3, a bioinformatics analysis using strand-specific RNA sequencing allowed the identification of 1113 potato long intergenic noncoding RNA (lincRNAs) from stem tissues. Long noncoding RNAs (lncRNAs) have been implicated in diverse regulatory roles in eukaryotes. Recently, defense-related lncRNAs have been identified in Arabidopsis and wheat. In this thesis we identified 559 potato lincRNAs that were differentially expressed (DE) in both cultivars compared to mock-inoculated controls, following inoculation by P. c. brasiliense. Furthermore, co-expression analysis associated 17 of these lincRNAs with 12 potato defense-related genes. These results suggest that lincRNAs possibly have functional roles in potato defence responses. Future work will focus on characterization of these lincRNAs in order to understand their specific functional roles, particularly in potato defense mechanisms. In chapter 4, regarding potential regulatory mechanisms employed by Pectobacterium species during survival under nutrient-limiting conditions, we described 137 sRNA transcripts in P. atrosepticum genome. About 62% of the identified sRNAs are conserved within the SRE. Furthermore, 68 sRNAs were differentially expressed when comparing P. atrosepticum cells under growth-promoting and starvation conditions; with 47 sRNAs up-regulated under nutrient-deficient conditions. Thus, since many starvation-induced sRNAs were identified, these findings highlighted that sRNAs play key roles in adaptive responses in the genus Pectobacterium.