Investigation on the role of plant defensin proteins in regulating plant-Verticillium longisporum interactions in Arabidopsis thaliana

Singh, Shailja

The hemibiotrophic pathogen Verticillium longisporum infects rapeseed (Brassica napus) and poses a real threat to its cultivation. Conventional approaches are limited and are based primarily on the availability of a wide gene pool to identify suitable genotypes for breeding in order to improve resistance, e.g. against abiotic stress or pathogens. In order to achieve this goal also in crop plants with a very narrow gene pool like rapeseed, new strategies have to be developed. One solution could be the expression of plant defensins (PDFs), which are known for their anti-fungal effects. In our laboratory, compatibility factors (CF) were identified in Arabidopsis, the loss of which led to a highly increased expression of AtPDF2.2. There are already numerous examples of how overexpression of such PDFs in model and crop plants led to improved and lasting resistance. This thesis work demonstrated the antifungal role of two AtPDF genes that contribute to the regulation of the Arabidopsis-Verticillium interaction. GUS expression under the AtPDF2.2 promoter showed that AtPDF2.2 was suppressed in Arabidopsis roots 6 days after infection (dpi) and later in leaves (9 dpi). This was confirmed by a transcript analysis that showed strong suppression of the AtPDF2.2 expression and two other Arabidopsis genes of PDF class II. Transgenic lines for AtPDF2.2 overexpression (OE-PDF2.2) and for knockdown (KD-pdf2.2) also showed an increased expression of AtPDF2.3 and AtPDF2.5 in the OE-PDF2.2 line, while a corresponding suppression in the KD-pdf2.2 line was recorded. Another important observation in the KD-pdf2.2 line was an enhanced jasmonic acid (JA) response, accompanied by upregulation of ethylene (ET) and salicylic acid (SA) -dependent genes, an effect that also in other infections with hemibiotrophic pathogens was observed. Therefore, based on available data, it can be assumed that V. longisporum uses host compatibility factors during the early phase of infection to suppress AtPDF2.2 to bypass plant defense. This also suppresses the expression of the closely related genes AtPDF2.3 and 2.5, which enables the fungus to establish a successful infection process. However, the suppression of these three PDFs, in turn, triggers the JA response which subsequently activates ET and SA signaling to mediate the reprogramming of defense response. To investigate the antifungal effect of AtPDF2.2, the OE-PDF2.2 line was analyzed, which showed increased resistance to two fungal (V. longisporum and Sclerotinia sclerotiorum) and one bacterial pathogen (Pseudomonas syringae). In contrast, the KD-pdf2.2 line showed a higher susceptibility to these fungal and bacterial pathogens. However, Verticillium longisporum induces the expression of AtPDF1.2a at 6 dpi in Arabidopsis wild type Col-0. Transcript study in the AtPDF1.2a overexpression (OE-PDF1.2a) and knockout (KO-pdf1.2a) plants revealed that AtPDF1.2a expression positively correlates with Class-I and III AtPDF genes, but negatively to AtPDF2.2, 2.3 and 2.5. Moreover, OE-PDF1.2a plants did not upregulate the expression of JA, ET or SA marker genes but KO-pdf1.2a plants showed induced expression of all the three pathways responsive genes. Possibly, low expression of AtPDF1.2a in KO-pdf1.2a plants during ongoing infection works as a concentration-specific signal. Since OE-PDF1.2a plants did not show significant changes in the expression of upstream JA, ET, and SA responsive genes it might rather be that upon V. longisporum attack AtPDF1.2a positively co-regulates several AtPDF genes from Class-I and III, while suppressing the expression of AtPDF2.2, 2.3 and 2.5 to reprogram the plant defense response. The antifungal activity has already been demonstrated for AtPDF1.2a, but its exact role in plant defense is still unclear. Therefore, OE-PDF1.2a lines were used, AtPDF1.2a seems to have an antifungal effect only on fungal pathogens; V. longisporum and Sclerotinia sclerotiorum but not on the bacterial pathogen.


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Singh, Shailja: Investigation on the role of plant defensin proteins in regulating plant-Verticillium longisporum interactions in Arabidopsis thaliana. 2020.


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