Small RNA profiling of virus-infected apple plants

Visser, Marike (2014-12)

Thesis (PhD)--Stellenbosch University, 2014.

Thesis

ENGLISH ABSTRACT: Apple stem grooving virus (ASGV) is globally associated with latent infection in commercial apple trees. Little is known about this plant-­‐virus interaction. This study made use of next-­‐generation sequencing to investigate the effect of virus-­‐infection on the expression of the different small RNA (sRNA) species namely, miRNAs, nat-­‐siRNAs, phasiRNAs, rasiRNAs, tRNA-­‐derived sRNAs and vsiRNAs. Broad and narrow size-­‐range datasets were generated using sRNA libraries prepared from total and size-­‐selected RNA, respectively. Through bioinformatic data analyses, 130 genomic loci were predicted to give rise to miRNAs, 85 of which were novel MIR genes. Targets were predicted for the majority of miRNAs, a few of which could be validated with a publicly available degradome dataset. Cis-­‐ and trans-­‐natural antisense transcripts (NATs) were identified, of which only the latter were highly enriched for sRNAs in their overlapping regions. Transcript as well as genomic regions were also identified that can give rise to phasiRNAs. For 25 of these loci an in-­‐phase miRNA target site was identified, half of which could be validated with the degradome dataset. Nearly all apple repeat sequences in Repbase were associated with sRNA synthesis. sRNAs derived from both ends of mature tRNAs were identified. These sRNAs corresponded to tRFs and tRNA-­‐halves. Reads associated with tRNA-­‐halves were prominent in the broad range datasets. sRNAs, originating from the central regions of tRNAs, were also observed. Analysis of the vsiRNAs suggested the presence of two ASGV genetic variants in two of the samples, while the third sample was infected with only one variant. Comparison of the vsiRNA profiles generated from the two datasets highlighted the influence of library preparation on the interpretation of results. Differential expression analysis of the identified apple sRNA species showed no variation between healthy and infected plants, except for the tRNA-­‐derived sRNAs, which did show altered expression levels. Taken together, the various sRNA species characterised in this study significantly extended the existing knowledge of apple sRNAs and provide a broad platform for future functional studies in apple. This study also presents the first and most comprehensive report on sRNAs involved in ASGV infection in apple.

AFRIKAANSE OPSOMMING: Appel gleufstam virus (ASGV) word wêreldwyd geassosieer met latente infeksie in kommersiële appelbome. Min inligting oor hierdie plant-­‐virus interaksie is beskikbaar. Hierdie studie het van volgende-­‐generasie volgordebepaling gebruik gemaak om die effek van virusinfeksie op die uitdrukking van verskillende klein RNA (sRNA) spesies, nl. miRNAs, nat-­‐siRNAs, phasiRNAs, rasiRNAs, tRNA-­‐afkomstige sRNAs en vsiRNAs, te ondersoek. Datastelle met breë en smal grootte-­‐verspreiding is gegenereer m.b.v. sRNA biblioteke wat onderskeidelik voorberei is vanaf totale RNA en RNA van ‘n bepaalde grootte. Deur middel van bioinformatiese data-­‐ontleding is 130 genomiese loci voorspel wat aanleiding kan gee tot miRNAs, waarvan 85 nuwe MIR gene is. Teikens is voorspel vir die meerderheid van die miRNAs en 'n aantal daarvan kon bevestig word m.b.v. 'n publiek-­‐beskikbare degradoom datastel. Cis-­‐ en trans-­‐natuurlike antisense transkripte (NATs) is geïdentifiseer, waarvan slegs die laasgenoemde verryk was vir sRNAs in hul oorvleuelende areas. Transkrip sowel as genomiese areas, wat aanleiding kan gee tot phasiRNAs, is ook geïdentifiseer. Vir 25 van hierdie loci is 'n in-­‐fase miRNA teiken geïdentifiseer, waarvan die helfte bevestig kon word met die degradoom datastel. Byna al die appel herhalende volgordes in Repbase was geassosieer met sRNA sintese. sRNAs afkomstig van beide kante van volwasse tRNAs is geïdentifiseer. Hierdie sRNAs het ooreengestem met tRFs en tRNA-­‐helftes. Volgordes geassosieer met tRNA-­‐helftes was prominent in die breë grootte-­‐verspreiding datastelle. sRNAs, afkomstig van die sentrale dele van tRNAs, is ook waargeneem. Ontleding van die vsiRNAs het die teenwoordigheid van twee ASGV genetiese variante in twee van die monsters aangetoon, terwyl die derde monster met slegs een variant geïnfekteer was. Die vergelyking van vsiRNA profiele, gegenereer vanaf die twee datasteltipes, beklemtoon die invloed van biblioteek voorbereiding op die interpretasie van resultate. Ontleding van die differensiële uitdrukking van die geïdentifiseerde appel sRNA spesies het geen verskil tussen gesonde en geïnfekteerde plante getoon nie, behalwe vir die tRNA-­‐afkomstige sRNAs, wat wel verandering die vlak van uitdrukking getoon het. Die verskillende sRNA spesies wat in hierdie studie geïdentifiseer is, het die bestaande kennis van appel sRNAs aansienlik uitgebrei en bied 'n breë platform vir toekomstige funksionele studies in appel. Hierdie studie bied ook die eerste, en mees omvattende verslag oor sRNAs betrokke in ASGV infeksie in appel.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/95828
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