Written by Bhanupratap Reddy Vanga at the New Zealand Institute of Plant and Food Research, University of Otago. This is the report from a BSPP Junior Fellowship. Click here to read more/apply for one yourself.
My BSPP Junior Fellowship award enabled me to visit Prof Ian Toth’s laboratory at James Hutton Institute (JHI), Dundee at a critical stage in my PhD studies to identify differential transcription of genes in the plant pathogen Pectobacterium atrosepticum (Pba) and a mutant (topoisomerase mutant) with increased recombination and reduced virulence on potatoes.
Pba is the causative agent of soft rot disease in potato tubers and blackleg disease in stems of potato plants. Horizontally Acquired Island 2 (HAI2), one of seventeen putative HAIs identified on the chromosome of Pba SCRI1043 encodes a virulence factor coronafacic acid (Cfa) that is required for pathogenicity on potato plants. HAI2 appears to be highly similar to a family of integrative and conjugative elements, which are acquired by horizontal gene transfer. The functions of most genes on the syntenic core of these elements are still unknown, but are likely to be involved in chromosomal excision, horizontal transfer and integration. As part of my PhD, we confirmed excision of HAI2 from the genome of Pba SCRI1043 that results in production of a circular episome. However, disruption of several genes belonging to the syntenic core of HAI2 altered the detectable level of HAI2 excision in media and/or in planta. Most notably, a mutant (ECAΔ0525) in ECA0525 increased the frequency of HAI2 excision from approximately 10-5 copies/cell in the wild-type to almost 10-1 copies /cell in the mutant during growth in culture.
ECA0525 (topBβ) is located on HAI2 and encodes a type IA topoisomerase, known as PbTopo IIIβ that belongs to the DNA Topoisomerase III. In pathogenicity tests on potato, the emergence of blackleg symptoms caused by Pba was reduced in plants inoculated with the PbTopo IIIβ mutant compared with plants exposed to the wild type. From these results, we hypothesized that the reduction in virulence of the PbTopo IIIβ mutant was likely due to changes in the transcription of the Cfa biosynthetic cluster resulting from topological changes in the island. However, quantitative PCR indicated that Cfa expression was not affected and that PbTopo IIIβ may control as yet unidentified genes involved in virulence of Pba SCRI1043. Therefore, while at JHI we investigated the effect of the PbTopo IIIβ mutant on genome-wide expression in Pba SCRI1043 by conducting Pba specific microarray experiments using facilities only available at JHI in an attempt to identify other factors that could be responsible for the reduced virulence.
Firstly, to identify the growth phase or time point when expression of topBβ was greatest and thus isolation of RNA was most appropriate for microarray experiments, I studied the transcription profile of topBβ at different growth stages. In this experiment, Pba SCRI1043 was grown in vitro and in planta for 64 hours (until late-stationary phase). RNA was isolated at 4 hours post inoculation (hpi), 12hpi, 20hpi, 40hpi and 64hpi for tubers and at every 8 hour interval for in vitro cultures until late-stationary phase. The amount of topBβ transcript at different time points was then investigated using qRT-PCR. Interestingly, the transcription of topBβ was unchanged throughout growth in media and in tubers. Therefore, we chose to isolate RNA for microarray experiments in early to mid-log phase from in vitro and in planta cultures.
For microarray analysis, the JHI provided technical support to process “two-coloured” Pba Agilent microarrays and expertise in microarray data analysis. The Agilent microarray contains 15000 Pba SCRI1043 probes and allowed the simultaneous analysis of 4 biological replicates. While I was at JHI, unfortunately, we couldn’t label the Pba SCRI1043 RNA with both Cy3 and Cy5 dye esters due to technical issues raised during labelling. As a result I re-isolated RNA and handed over samples for microarray processing prior to returning to New Zealand. These microarray results will be re-confirmed by targeted qRT-PCR analysis, once we obtain the microarray results.
The 5- week visit was a valuable experience for me to gain knowledge regarding microarray processing, data analysis and other related molecular techniques. This visit also played a critical role in development of a strong collaborative relationship between New Zealand Institute of Plant and Food Research, JHI and University of Otago from which a scientific publication is imminent. During my stay in Scotland, I also enjoyed visiting beautiful Highlands, Edinburgh and my previous academic supervisors at University of Glasgow and JHI. I would like to thank Prof Ian Toth, Dr Pete Hedly, Dr Sonia Humphris, Dr Emma Campbell and Dr Jenny Morris for hosting me at JHI and for taking their own time out to supervise my work, ensuring that my visit was not only scientifically productive but also a great personal experience. I would also like to thank my supervisors in New Zealand, Dr Andrew Pitman and Prof Clive Ronson, for their commitment and support that made this visit possible. Finally, many thanks to the BSPP for providing the funds for such a productive trip.
Bhanupratap Reddy Vanga
New Zealand Institute of Plant and Food Research,
University of Otago.