The adaptive evolution of the plant pathogen Albugo candida

Albugo candida is a plant pathogen that has been reported on many host species. While multiple host-specific races have long been recognized in A. candida, the genetic variation of these races has never been explored in nature and little is known about how the pathogen has adapted to its many hosts. Recently, evidence of genetic exchanges between races suggested that hybridization played an important role in the evolution of A. candida races. The authors also demonstrated that host-specific races of A. candida can co-occur, provided the immune system of the host is compromised by a compatible race. This immunosuppression by A. candida had previously been shown to allow growth of other pathogens. To study both these phenomena (the evolution of and the host immunosuppression imposed by A. candida), a capture array was designed to sequence 187 loci (~660,000 bp) from A. candida and loci from 47 other plant pathogens. In Chapter 3, I explain the rationale and methodology behind this approach. I show that it is cost-effective and that it may be used to identify microorganisms directly from a leaf and make inference about pathogen abundance within samples. In Chapter 4, genetic diversity of A. candida is analysed at a 400 kb contig and 32 diversity-tracking genes. Races are identified based on genetic divergence and recombination is investigated within and between races. In Chapter 5, I investigate genetic diversity at heterozygous sites to study the ploidy level and the reproductive mode of A. candida races as well as to detect mixed A. candida infections and loss-of-heterozygosity events. In this thesis, I demonstrate that A. candida races adapt to their hosts using complex mechanisms and that some may, in the long term, speciate. I also provide a novel method which may be used to interrogate microbial diversity directly from the field

Identification of Avramr1 from Phytophthora infestans using long read and cDNA pathogen-enrichment sequencing (PenSeq)

Potato late blight, caused by the oomycete pathogen Phytophthora infestans, significantly hampers potato production. Recently, a new Resistance to Phytophthora infestans (Rpi) gene, Rpi‐amr1, was cloned from a wild Solanum species, Solanum americanum. Identification of the corresponding recognized effector (Avirulence or Avr) genes from P. infestans is key to elucidating their naturally occurring sequence variation, which in turn informs the potential durability of the cognate late blight resistance. To identify the P. infestans effector recognized by Rpi‐amr1, we screened available RXLR effector libraries and used long read and cDNA pathogen‐enrichment sequencing (PenSeq) on four P. infestans isolates to explore the untested effectors. Using single‐molecule real‐time sequencing (SMRT) and cDNA PenSeq, we identified 47 highly expressed effectors from P. infestans, including PITG_07569, which triggers a highly specific cell death response when transiently coexpressed with Rpi‐amr1 in Nicotiana benthamiana, suggesting that PITG_07569 is Avramr1. Here we demonstrate that long read and cDNA PenSeq enables the identification of full‐length RXLR effector families and their expression profile. This study has revealed key insights into the evolution and polymorphism of a complex RXLR effector family that is associated with the recognition by Rpi‐amr1

Pathogen enrichment sequencing (PenSeq) enables population genomic studies in oomycetes

The oomycete pathogens Phytophthora infestans and P. capsici cause significant crop losses world‐wide, threatening food security. In each case, pathogenicity factors, called RXLR effectors, contribute to virulence. Some RXLRs are perceived by resistance proteins to trigger host immunity, but our understanding of the demographic processes and adaptive evolution of pathogen virulence remains poor. Here, we describe PenSeq, a highly efficient enrichment sequencing approach for genes encoding pathogenicity determinants which, as shown for the infamous potato blight pathogen Phytophthora infestans, make up < 1% of the entire genome. PenSeq facilitates the characterization of allelic diversity in pathogen effectors, enabling evolutionary and population genomic analyses of Phytophthora species. Furthermore, PenSeq enables the massively parallel identification of presence/absence variations and sequence polymorphisms in key pathogen genes, which is a prerequisite for the efficient deployment of host resistance genes. PenSeq represents a cost‐effective alternative to whole‐genome sequencing and addresses crucial limitations of current plant pathogen population studies, which are often based on selectively neutral markers and consequently have limited utility in the analysis of adaptive evolution. The approach can be adapted to diverse microbes and pathogens

Data from: The effects of recombination, mutation and selection on the evolution of the Rp1 resistance genes in grasses

Plant immune genes, or resistance genes, are involved in a co-evolutionary arms race with a diverse range of pathogens. In agronomically important grasses, such R genes have been extensively studied because of their role in pathogen resistance and in the breeding of resistant cultivars. In this study, we evaluate the importance of recombination, mutation and selection on the evolution of the R gene complex Rp1 of Sorghum, Triticum, Brachypodium, Oryza and Zea. Analyses show that recombination is widespread, and we detected 73 independent instances of sequence exchange, involving on average 1567 of 4692 nucleotides analysed (33.4%). We were able to date 24 interspecific recombination events and found that four occurred postspeciation, which suggests that genetic introgression took place between different grass species. Other interspecific events seemed to have been maintained over long evolutionary time, suggesting the presence of balancing selection. Significant positive selection (i.e. a relative excess of nonsynonymous substitutions (dN/dS>1)) was detected in 17–95 codons (0.42–2.02%). Recombination was significantly associated with areas with high levels of polymorphism but not with an elevated dN/dS ratio. Finally, phylogenetic analyses show that recombination results in a general overestimation of the divergence time (mean = 14.3%) and an alteration of the gene tree topology if the tree is not calibrated. Given that the statistical power to detect recombination is determined by the level of polymorphism of the amplicon as well as the number of sequences analysed, it is likely that many studies have underestimated the importance of recombination relative to the mutation rate

Maximum clade credibility trees using the truncated alignment with no priors on divergence times.

Maximum clade credibility trees using the truncated alignment with no priors on divergence times. Trees were inferred using BEAST v1.62. Estimated ages of divergence are depicted in bold and posterior probabilities in italic. Abbreviations, Sb: Sorghum bicolor, Ta1 and Ta2: Triticum aestivum, Bd: Brachypodium distachyon

Maximum clade credibility trees using the total alignment as well as fossil calibrations

Maximum clade credibility trees using the total alignment as well as fossil calibrations. Trees were inferred using BEAST v1.62. Estimated ages of divergence are depicted in bold and posterior probabilities in italic. Abbreviations, Sb: Sorghum bicolor, Ta1 and Ta2: Triticum aestivum, Bd: Brachypodium distachyon

Maximum clade credibility trees using the total alignement with no priors on divergence times.

Maximum clade credibility trees using the total alignment with no priors on divergence times. Trees were inferred using BEAST v1.62. Estimated ages of divergence are depicted in bold and posterior probabilities in italic. Abbreviations, Sb: Sorghum bicolor, Ta1 and Ta2: Triticum aestivum, Bd: Brachypodium distachyon

Truncated alignment of Rp1 sequences from the 5 Poaceae genera used in this study.

This alignment was used to construct phylogenetic trees with and without calibration points and to test for a correlation between positively-selected amino-acids and the incidence of recombination

Total alignment of Rp1 sequences from the 5 Poaceae genera used in this study.

This alignment was used to construct phylogenetic trees with and without calibration points and evaluate the incidence of recombination