Epigenetic Gene Silencing of Virulence Effectors in the Plant Pathogen Phytophthora

Plant pathogens of the genus Phytophthora are noted for their destructiveness and adaptability. The rapid phenotypic diversification of clonal lineages in natural and laboratory settings has long puzzled scientists. The alternate loss and recovery of virulence in clonally propagated cultures has been observed for the soybean root rot pathogen Phytophthora sojae and the potato late blight pathogen Phytophthora infestans. We have studied the structure, expression, and inheritance of avirulence effector genes in P. sojae. By performing outcrosses between naturally occurring strains of P. sojae, we demonstrate that transgenerational gene silencing of Avr3a is allele-specific and associated with small RNA. Bisulphite sequencing of the Avr3a locus in numerous P. sojae strains did not result in any evidence for cytosine methylation. We failed to detect methylated DNA in P. sojae using antibodies to 5-methyl cytosine, whereas this was readily detected in soybean DNA. These results support suggestions that P. sojae does not have the capability to methylate DNA, and that epigenetic gene silencing must rely on other mechanisms. Treatment of cultures indicates that silencing of Avr3a is unresponsive to the DNA methylation inhibitor 5-aza-2-deoxycytidine (50 uM), the histone H3K9 methyltransferase inhibitor BIX-01294 (5 uM), and to the histone deacetylase inhibitors trichostatin A (10 uM) and butyric acid (5 mM). Tracking progeny shows that transgenerational gene silencing of Avr3a is exceptionally stable, but test crosses indicate that silencing can be released by outcrossing.  Overall, our results suggest strain-specific interplay between conventional- and epi-genetic variation in Phytophthora.