Single Amino Acid Substitution the DNA Repairing Gene Radiation-Sensitive 4 Contributes to Ultraviolet Tolerance of a Plant Pathogen

Abstract

To successfully survive and reproduce, all species constantly modify the structure and expression of their genomes to cope with changing environmental conditions including ultraviolet (UV) radiation. Thus, knowledge of species adaptation to environmental changes is a central theme of evolutionary studies which could have important implication for disease management and social-ecological sustainability in the future but is generally insufficient. Here, we investigated the evolution of UV adaptation in organisms by population genetic analysis of sequence structure, physiochemistry, transcription, and fitness variation in the radiation-sensitive 4 (RAD4) gene of the Irish potato famine pathogen Phytophthora infestans sampled from various altitudes. We found that RAD4 is a key gene determining the resistance of the pathogen to UV stress as indicated by strong phenotype-genotype-geography associations and upregulated transcription after UV exposure. We also found conserved evolution in the RAD4 gene. Only five nucleotide haplotypes corresponding to three protein isoforms generated by point mutations were detected in the 140 sequences analyzed and the mutations were constrained to the N-terminal domain of the protein. Physiochemical changes associated with non-synonymous mutations generate severe fitness penalty to mutants, which are purged out by natural selection, leading to the conserved evolution observed in the gene.

Keywords: UV adaptation; agriculture; climate change; evolutionary ecology; natural selection; nucleotide excision repair system; population genetics; transcriptional regulation.

FIGURE 1

Gene and protein structure of Phytophthora infestans RAD4. (A) Nucleotide sequences including introns of the five haplotypes detected. Dots indicate identical nucleotides to the reference sequence, defined as Haplotype 1 (H1). (B) Deduced amino acid sequences in the isoforms (Iso). Different amino acids in the sequences were shown in white and shared amino acids were showed in red. (C) Protein structure predicted by IUPred2A using the reference sequence downloaded from NCBI (assessment number: 9474425).

FIGURE 2

Nucleotide haplotype network of RAD4 gene in the Phytophthora infestans populations sampled from seven production area in China. Each circle represents a unique haplotype, and the size of circles represents the proportion of the haplotypes in the sample pooled from the seven locations. The short-term marks represent steps of nucleotide substitution between two haplotypes.

FIGURE 3

Hydrophilic estimate and structure prediction of Isoform-1: (A) Hydrophobicity estimated by a Kyte & Doolittle (K-D) approach embedded in the BioEdit program; and (B) intrinsically disordered protein regions (IDPRs) estimated by MobiDB.

FIGURE 4

Solvent accessibility of three RAD4 isoforms. A system of neural networks with a window size of 17 was used to predict the solvent accessibility.

FIGURE 5

Quantitative real-time PCR analysis of RAD4 expression. The qPCR was carried out by quantifying the expression of RAD4 relative to the Actin A housekeeping gene using the 2-ΔΔCT method. The experimental isolates (A–D) were either exposed to 20-min UVC radiation in each of for 8-day period or without UV treatment (CK). ^∗Indicates a significant difference (p < 0.05) between UV treated and non-treated isolates in RAD4 expression.