The study of transcriptional regulation of necrotrophic effector genes ToxA and Tox3 in the wheat pathogen Parastagonospora nodorum
Creators: name: Lin, Shao-Yu
Publisher: name: The Australian National University
Year: 2017
Subjects: Parastagonospora nodorum
necrotrophic effector
ToxA
Tox3
wheat
transcription factor
PnCon7
PnPf2
transcriptional regulation
alternative splicing
pathogen
infection
plant
pathogenicity
fungus

description: Parastagonospora nodorum (P. nodorum) is an important necrotrophic fungus on wheat causing significant yield losses in Australia per annum. It is now widely recognized that P. nodorum causes disease by secreting necrotrophic effectors (NEs) which induce necrosis on susceptible wheat leaves through an inverse gene for gene manner. Two of the NEs described are ToxA and Tox3. Previous reports have shown that ToxA and Tox3 are highly upregulated during infection. However, how these effectors are regulated and expressed is largely unclear. To better understand the transcriptional regulation of ToxA and Tox3, I conducted functional analyses on their promoter regions to identify the key factors for the regulation in P. nodorum. Being a necrotroph, P. nodorum tends to acquire nutrients from surrounding environment. It is therefore hypothesized that P. nodorum senses nutrients during infection to trigger the expression of the NE genes. By analyzing the ability of promoters to trigger gene expression under different nutrient condition, it was found that nitrogen sources such as nitrate and ammonium involved in expression of Tox3. On the other hand, the expression of ToxA was extremely weak in all tested media. Moreover, through promoter deletion analysis, I was unable to identify a cis-regulatory element other than TATA box on the ToxA promoter. DNase I footprinting and yeast one-hybrid assay also did not identify protein candidates interacting with the ToxA promoter. Interestingly, the ToxA promoter showed ability to induce strong expression in the tested media after replacing the ToxA gene with GFP. In contrast, much lower GFP transcripts were observed in the presence of ToxA CDS when expressed under the same ToxA promoter. These results imply that ToxA may be regulated at transcriptional elongation or post-transcriptional stage. In previous studies, it is recognized that Tox3 was regulated by transcription factors. Nevertheless, none of the studies showed evidence of direct interaction. In this study, the promoter and expression of Tox3 was characterized. A combination of promoter deletion and DNase I footprinting experiments identified a 25 bp region (Tox3-URE1) on the Tox3 promoter as being required for transcription. Subsequent yeast one-hybrid analysis identified a C2H2 zinc finger transcription factor PnCon7 specifically interacting with Tox3-URE1. Silencing of PnCon7 resulted in the down-regulation of Tox3 in a dose dependent manner. Moreover, pathogenicity and expressions of two other necrotrophic effectors (ToxA and Tox1) were identified to be affected in an indirect way. Collectively, in my PhD study, I did not identify cis-regulatory elements or transcription factors responsible for the expression of ToxA. Alternatively, ToxA was found to be regulated at transcriptional elongation or post-transcriptional stage. On the other hand, the expression of Tox3 was largely upregulated when encountering specific nitrogen source. Furthermore, a C2H2 zinc finger transcription factor PnCon7 was found to specifically bind to the Tox3 promoter and regulated its expression through a dose dependent manner. This study highlights the latest understanding of how NEs are regulated at the transcriptional level in fungi and provides a fundamental research for future studies in transcriptional regulation of fungal virulence genes.