Transcriptomic studies of the early stages of potato infection by Phytophthora infestans
The late blight pathogen, Phytophthora infestans, is the most destructive pathogen of its solanaceous hosts potato and tomato. It is a threat to global food security and it is therefore important to understand the cellular and molecular dynamics underlying colonisation of its host plants. This greater understanding will inform strategies to improve host plant resistance. In addition to studying the cell biology of the interaction, it is important to understand the temporal changes in gene expression and regulation during host-pathogen interactions at the earliest infection time points. Previously published transcriptomic studies of P. infestans used two days post infection (dpi) as the earliest sampling time point. Expression of a marker gene (Hmp1) for biotrophy and a selection of effector coding genes has been reported as early as 12 hours post inoculation (hpi), suggesting that infection was initiated before then. Transcriptomic studies of P. infestans have focussed mostly on leaf tissue, and there is still a lack of research on the transcriptome of P. infestans grown in alternative plant tissues such as tubers, or in host cell-free apoplastic fluid. This thesis explores transcriptomic studies of the early, biotrophic stages of potato infection by Phytophthora infestans, which is critical for understanding which genes are involved at what stages of infection development. By using the latest sensitive microarray technology to study the P. infestans transcriptome in an infection time course that remained biotrophic for its duration, a list of 1,707 transcripts of P. infestans were discovered to be differentially expressed. This list included 114 transcripts for RxLR effectors, out of which 26 were detected from 12 hours post infection, including: Avr2, Avr3a, Avrblb1 (ipi01), Avrblb2, and the recently characterised RD2. Also of interest was that transcripts encoding a PAMP (CBEL) detected at 12 hours, were suppressed in the pathogen by 24 hours. Transcripts encoding 55 RxLR effectors were co-expressed (with >95 % correlation coefficient) with the biotrophy marker gene Hmp1, suggesting that these effectors are important throughout the biotrophic stages of infection. QRT-PCR and cell biology data supported the expression of the biotrophy marker gene Hmp1 as early as 12 hours after infection and this was further supported by the co-expression of avirulence genes such as Avr2 and Avr3a. A set of 17 transcripts, including six cytoplasmic effectors (RxLR effectors), as well as a transcript encoding an apoplastic effector (glucanase inhibitor), was found to be infection-specific, supporting the hypothesis that these genes might have roles in establishing biotrophy. By examining pathogen behaviour in tuber tissue, clear cell biology evidence of functional haustoria was found. Gene expression analysis of a selection of leaf infection-related genes suggested that effectors are used to promote infection also in host tuber tissue. However, some cytoplasmic RXLR effector proteins such as PITG_05146 and PITG_15128, which were up-regulated during biotrophic infection of leaf tissue, were not detected during tuber infection, indicating potential differences in pathogenic requirements. A microarray experiment was conducted on in vitro stages of zoospores, and mycelium grown in apoplastic fluid of N. benthamiana, nutrient rich pea broth, and sterile water. This revealed 13,819 transcripts that were differentially expressed between any two conditions. This list included transcripts encoding 322 RxLR effectors, of which avirulence effectors such as Avr2, Avr3a, and RD2 were highly up-regulated during hyphal growth in apoplastic fluid compared to other in vitro stages. This provides evidence that the apoplast contains chemical signals that induce expression of infection-related genes in P. infestans. Curiously, the leaf infection-specific genes identified in Chapter 3 were not expressed when P. infestans was grown in apoplastic fluid, revealing that additional stimuli are required for induction of all necessary pathogen genes during infection. Future research, building upon the findings from this project, should be focused on the following areas: 1) Explore whether haustoria are produced only in order to deliver effectors or if there are other purposes as well, such as nutrient uptake; 2) The continued exploration of differences between genes co-expressed with Hmp1 during leaf infection, tuber infection, and in apoplastic fluid to further dissect the transcriptional regulation of these genes; 3) Identify whether Hmp1-co-expressed genes of unknown function may play a role in haustorium formation; 4) Investigate, using molecular transformation and cell biology, whether secreted proteins co-expressed with Hmp1 are secreted from haustoria; 5) Investigate the role(s) of infection-specific genes in establishing disease. 6) Transcriptomic studies of P. infestans biotrophic infection of tuber tissue to determine the differences in pathogenic adaptation in this tissue type, compared to leaf infection.
Phytophthora infestans, Transcriptome, Early potato infection
Networked Digital Library of Theses and Dissertations