Sequence diversity in the large subunit of RNA polymerase I contributes to Mefenoxam insensitivity in Phytophthora infestans

Abstract

Phenylamide fungicides have been widely used for the control of oomycete-incited plant diseases for over 30 years. Insensitivity to this chemical class of fungicide was recorded early in its usage history, but the precise protein(s) conditioning insensitivity has proven difficult to determine. To determine the genetic basis of insensitivity and to inform strategies for the cloning of the gene(s) responsible, genetic crosses were established between Mefenoxam sensitive and intermediate insensitive isolates of Phytophthora infestans, the potato late blight pathogen. F1 progeny showed the expected semi-dominant phenotypes for Mefenoxam insensitivity and suggested the involvement of multiple loci, complicating the positional cloning of the gene(s) conditioning insensitivity to Mefenoxam. Instead, a candidate gene strategy was used, based on previous observations that the primary effect of phenylamide compounds is to inhibit ribosomal RNA synthesis. The subunits of RNA polymerase I (RNApolI) were sequenced from sensitive and insensitive isolates and F1 progeny. Single nucleotide polymorphisms (SNPs) specific to insensitive field isolates were identified in the gene encoding the large subunit of RNApolI. In a survey of field isolates, SNP T1145A (Y382F) showed an 86% association with Mefenoxam insensitivity. Isolates not showing this association belonged predominantly to one P. infestans genotype. The transfer of the 'insensitive' allele of RPA190 to a sensitive isolate yielded transgenic lines that were insensitive to Mefenoxam. These results demonstrate that sequence variation in RPA190 contributes to insensitivity to Mefenoxam in P. infestans.

Keywords: Mefenoxam; Metalaxyl; Phytophthora infestans; RNA polymerase; fungicide; oomycete; phenylamide.

Figure 1

Growth of Phytophthora infestans  F1 progeny from a cross of 88133 × T30‐4 on rye agar amended with 5 μg/mL (a) and 100 μg/mL (b) Mefenoxam. Growth on Mefenoxam‐amended medium is presented as a percentage of growth of the culture on medium containing no Mefenoxam (y axis). Parental strains and F1 progeny are shown on the x axis. Parental isolate 88133 is fully sensitive to Mefenoxam, whereas the T30‐4 parental isolate is intermediate insensitive. Isolate 80029, a parent of T30‐4, is included here for comparison. Data are the mean of 12 measurements; standard deviation from the mean is shown for each P. infestans line.

Figure 2

Organization of predicted functional domains within Phytophthora infestans  RPA190, showing the location of single nucleotide polymorphisms (SNPs) identified from 15 isolates (shown to scale). The RPA190 gene sequence is shown as a solid black line. Regions encoding the functional domains of RPA190 are shown above the gene sequence: clamp (purple), active site (yellow), pore (blue), funnel (pink) and cleft (green). SNPs identified from 15 P. infestans isolates are shown as short vertical black bars above the solid black line of the gene; a double vertical bar represents two SNPs within a single codon. SNPs leading to changes in the amino acid sequence of RPA190 are shown under the solid black line of the gene; pink bars represent amino acid substitutions found only in isolates that are insensitive to Mefenoxam. T1145A (Y382F) is indicated by a black triangle.

Figure 3

Transfer of the RPA190 allele from an insensitive isolate of Phytophthora infestans to sensitive isolate Ca65. Wild‐type Ca65 (top) exhibits only minimal aerial growth at a high dose (100 μg/mL). Transgenic lines POLI‐60‐2 and POLI‐1–4 grow slowly in the absence of Mefenoxam, but continue to grow into the growth medium at 100 μg/mL of Mefenoxam. Image was taken after 12 days of growth in 90‐mm‐diameter Petri dishes.