Powdery mildew, a fungal disease of plants, is one of the most significant causes of crop disease and yield loss worldwide. Collectively, this group of pathogens infects a diverse set of plant hosts, including wheat, barley, grape, and ornamental species. One such mildew, Golovinomyces cichoracearum, is the causative agent of powdery mildew disease on susceptible cucurbit species. The ability of this mildew to infect the model plant Arabidopsis thaliana has allowed for the study of the biology of this important class of plant pathogens in a laboratory setting.
When exposed to a susceptible host, G. cichoracearum forms a feeding structure within the plant cell, known as a haustorium. The fungus initiates changes in plant cell structure, gene expression, and nutrient transport to allow for its survival and reproduction. Relatively little is known about the molecular and cellular mechanisms employed by the fungus to elicit these cellular changes and evade the plant immune response. This is partially due to the recalcitrance of the fungus to genetic manipulation, the large and complicated nature of the powdery mildew genome, and the lack of genetic and genomic tools that have been developed for the study of this class of organisms.
Here, we describe the development of an Agrobacterium tumefaciens-mediated tool for transiently silencing G. cichoracearum genes during infection of Arabidopsis. We demonstrate that this technique can be successfully employed at the early stages of powdery mildew infection, and that silencing an essential fungal gene, GcCYP51, results in reduced haustorial formation and subsequent fungal growth. We then use this technique to identify three G. cichoracearum effectors, GcEC8, GcEC10, and GcEC17, that are required for virulence on Arabidopsis.
We then describe efforts to characterize these effectors in terms of gene expression, sub-cellular localization, the identification of plant interacting partners, bioinformatic prediction, and their roles in the suppression of the plant immune response.
GcEC10 is characterized as an RNase-like protein and is localized in the plant cytosol and nucleus. GcEC10 may interact with Arabidopsis proteins AtEDR4 and AtPHOS32, two proteins implicated in the plant immune response to pathogens. We determined that GcEC10 expression suppresses the hypersensitive response (HR) elicited by the plant resistance gene/effector pair Bs2/AvrBs2. We propose a potential model in which GcEC10 attenuates the plant immune response leading to the hypersensitive response and immunity to powdery mildew via interference with mitogen-activated protein kinase cascades associated with plant immunity.
GcEC8 shares no sequence or domain homology with any proteins outside of the powdery mildews, and may interact with the plant protein AXR3, an auxin-responsive transcription factor. We propose that GcEC8 may interfere with the plant auxin response, leading to increased disease resistance, although the specific mechanism is not yet known.
GcEC17 is a highly-conserved powdery mildew effector, and is also characterized as an RNase-like protein. GcEc17 is localized in the plant nucleus and cytosol. Two hypothetical Arabidopsis genes, At4g29905 and At3g32930, were predicted to interact with GcEC17 via yeast two-hybrid, however we have not yet conceived of a model for GcEC17 action during powdery mildew infection.
We further describe efforts towards transient plasmid transformation of G. cichoracearum, which we hope will eventually lead to the ability of researchers to introduce the Cas9 genome editing system into the fungus. This might allow for the creation of stable, targeted genetic mutants, which has not yet been achieved in any powdery mildew species. Our efforts were unsuccessful, and here we detail the attempted methodologies in the hopes that future researchers may find more success.
Finally, we describe a method developed to achieve the purification of high molecular weight genomic DNA from G. cichoracearum suitable for Pacific Biosciences Single Molecule Long-Read Sequencing, and the analysis of the genome sequence obtained using this method. We compare the genome of G. cichoracearum to the genomes of four other sequenced powdery mildew species, Blumeria graminis f. sp. hordei, B. graminis f. sp. hordei, Erysiphe pisi, and Golovinomyces orontii. We found that the genomes of the mildews are similar in size and gene content, however we found that each species encodes a large, unique repertoire of predicted effector proteins.
These experiments provide some of the first insights into the genome and effector biology of the enigmatic plant pathogen G. cichoracearum. By combining novel molecular techniques with next-generation sequencing approaches, we now have a more complete idea of the mechanisms by which this fungus causes disease on its host plants.