Modulation of the Vitis vinifera cv. ‘Chardonnay’ microRNA and mRNA transcriptomes in response to aster yellows phytoplasma-infection

Thesis (PhDAgric)--Stellenbosch University, 2019.A published article based on the PhD is available in this repository at http://hdl.handle.net/10019.1/102169ENGLISH ABSTRACT: Aster yellows (AY) phytoplasmas are part of a group of cell wall-less plant pathogenic bacteria responsible for a detrimental disease known as grapevine yellows (GY). The molecular mechanisms of AY phytoplasma pathogenicity on highly susceptible cultivars, such as Chardonnay, are still largely unknown. This has sparked considerable interest to gain knowledge about the basis of host susceptibility to GY in order to develop control strategies that may mitigate the scale of infection or even prevent spread. Leaf total RNA was extracted from both healthy and AY-infected plants to generate small RNA (sRNA) sequencing libraries, as well as mRNA sequencing libraries. These libraries were subjected to Illumina transcriptome sequencing (small RNA-seq and mRNA-seq, respectively), and comparative transcriptome profiling, to explore the involvement of microRNA (miRNA) and gene expression pathways in AY phytoplasma-infected Chardonnay. Multiple known miRNA sequence variants (isomiRs) were identified, and 13 known miRNAs were shown to be differentially expressed. A total of 175 novel miRNA precursor sequences, each derived from a previously uncharacterised genomic location, were identified, of which 23 were differentially expressed. Some of these novel miRNAs shared high sequence similarity with conserved miRNAs from other plant species, as well as known grapevine miRNAs. The relative expression of some of these known and novel miRNAs was confirmed with stem-loop RT-qPCR analysis, thereby validating the trend of miRNA expression in the normalised sRNA-seq read count data. miRNA target prediction, using a complementary-based in silico approach, followed by functional annotation, allowed the identification of potential target genes involved in plant morphology, hormone signalling, nutrient homeostasis, as well as plant stress. mRNA-seq results showed that 175 genes were differentially expressed in the AY phytoplasma-infected leaf material. Functional annotation of differentially expressed genes (DEGs) enabled the identification of mRNAs involved in plastid and cell wall metabolism/architecture, signalling, innate immunity, pathogen defence, secondary metabolism and photosynthesis. RT-qPCR analysis was used to validate the trend of expression of significant DEGs. Taken together, this study presents the first report on the modulation of miRNAs and genes associated with AY phytoplasma-infection in Chardonnay. The knowledge generated during this study may be crucial in understanding disease symptom development in AY phytoplasma-infected grapevines. Importantly, the findings of this study may also aid in developing GY disease control strategies and could provide added insight for future plant pathogenesis-related studies.AFRIKAANSE OPSOMMING: Fitoplasmas is selwandlose, patogeniese bakterieë waarvan die astervergelingfitoplasmas deel is. Astervergelingsfitoplasmas kan ‘n nadelige siekte in wingerd veroorsaak wat bekend staan as wingervergeling-siekte. Die onderliggende molekulêre meganismes betrokke by astervergelings-patogenisiteit in hoogsvatbare kultivars soos Chardonnay is meestal onbekend. Dit het gelei tot ‘n toenemende belangstelling om die basis van vatbaarheid deur die gasheerplant te begryp en sodoende voorkomingstrategieë teen die siekte te onwikkel. Blaar totale ‘RNA’ is geïsoleer uit gesonde, sowel as astervergelingsfitoplasma-geïnfekteerde plante, en gebruik om beide klein RNA (‘sRNA’) biblioteke asook boodskapper RNA (‘mRNA’) biblioteke te genereer vir volgende-generasie volgordebepaling. Illumina transkriptoom volgordebepaling (onderskeidelik ‘small RNA-seq’ en ‘mRNA-seq’) en relatiewe transkriptoom profielsamestellings is gebruik om die moontlike betrokkenheid van mikro-‘RNAs’ (‘miRNAs’) en gene in Chardonnay met wingervergeling-siekte te ondersoek. ‘n Groot aantal variante van bekende miRNAs (‘isomiRs’) is geïdentifiseer en daar is ook vasgestel dat 13 reeds bekende miRNAs differensieël uitgedruk is. 175 ‘nuwe’ miRNA volgordes is ook geïdentifiseer binne onbekende genoomareas waarvan 23 differensieël uitgedruk is. Nukleotiedvolgorde-analises is uitgevoer om eendersheid tussen hierdie ‘nuwe’ miRNAs en reeds bekende wingerd miRNAs asook ander plant miRNAs te bewys. ‘n Stam-lus tru-transkripsie kwantitatiewe polimerase kettingreaksie (‘stem-loop RT-qPCR’) metode is ingespan om die uitdrukkingspatrone van bekende en ‘nuwe’ miRNAs te beklemtoon. miRNA teikens is bepaal deur middel van ‘n komplementêr-gebaseerderde in silico metode. Daaropvolgende funksiebepalings het miRNA teikens geïdentifiseer wat betrokke is in plantmorfologie, hormoonregulering, voedingstofregulering, en plantstres. Die gebruik van ‘mRNA-seq’ het miljoene goeie-kwaliteit volgordes verskaf wat gebruik is om differensieële geenuitdrukking te bepaal. 175 differensieël uigedrukte gene (‘DEGs’) is deur middel van hierdie metode in the geïnfekteerde blaarmateriaal geïdentifiseer. Funksiebepalings het gewys dat die ‘DEGs’ verskillende rolle het in byvoorbeeld plastied- en selwandontwikkeling, boodskapverspreiding, immuniteit, patogeenbeskerming, sekondêre metabolisme en fotosintese. ‘RT-qPCR’ analise is gebruik om die uitdrukkingspatrone van die beduidende ‘DEGs’ te bevestig. Die bestaande studie is die eerste navorsingsprojek wat die identifisering en uitdrukkingsanalise van miRNAs en gene in astervergelingsfitoplasma-geïnfekteerde Chardonnay ondersoek. Die bogenoemde bevindinge kan gebruik word om kennis met betrekking tot simptoomontwikkeling weens wingerdvergeling-siekte te verbreed. Hierdie studie kan ook nuwe geleenthede en motiverings skep vir verdere diepgaande studies in hierdie veld en kan moontlik toegepas word in pogings om wingervergeling-siekteweerstand te bewerkstellig