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.

Albugo candida race diversity, ploidy and host-associated microbes revealed using DNA sequence capture on diseased plants in the field.

Physiological races of the oomycete Albugo candida are biotrophic pathogens of diverse plant species, primarily the Brassicaceae, and cause infections that suppress host immunity to other pathogens. However, A. candida race diversity and the consequences of host immunosuppression are poorly understood in the field. We report a method that enables sequencing of DNA of plant pathogens and plant-associated microbes directly from field samples (Pathogen Enrichment Sequencing: PenSeq). We apply this method to explore race diversity in A. candida and to detect A. candida-associated microbes in the field (91 A. candida-infected plants). We show with unprecedented resolution that each host plant species supports colonization by one of 17 distinct phylogenetic lineages, each with an unique repertoire of effector candidate alleles. These data reveal the crucial role of sexual and asexual reproduction, polyploidy and host domestication in A. candida specialization on distinct plant species. Our bait design also enabled phylogenetic assignment of DNA sequences from bacteria and fungi from plants in the field. This paper shows that targeted sequencing has a great potential for the study of pathogen populations while they are colonizing their hosts. This method could be applied to other microbes, especially to those that cannot be cultured.

Random mutagenesis screen shows that Phytophthora capsici CRN83_152-mediated cell death is not required for its virulence function(s).

With the increasing availability of plant pathogen genomes, secreted proteins that aid infection (effectors) have emerged as key factors that help to govern plant-microbe interactions. The conserved CRN (CRinkling and Necrosis) effector family was first described in oomycetes by their capacity to induce host cell death. Despite recent advances towards the elucidation of CRN virulence functions, the relevance of CRN-induced cell death remains unclear. In planta over-expression of PcCRN83_152, a CRN effector from Phytophthora capsici, causes host cell death and boosts P. capsici virulence. We used these features to ask whether PcCRN83_152-induced cell death is linked to its virulence function. By randomly mutating this effector, we generated PcCRN83_152 variants with no cell death (NCD) phenotypes, which were subsequently tested for activity towards enhanced virulence. We showed that a subset of PcCRN83_152 NCD variants retained their ability to boost P. capsici virulence. Moreover, NCD variants were shown to have a suppressive effect on PcCRN83_152-mediated cell death. Our work shows that PcCRN83_152-induced cell death and virulence function can be separated. Moreover, if these findings hold true for other cell death-inducing CRN effectors, this work, in turn, will provide a framework for studies aimed at unveiling the virulence functions of these effectors.

A Perspective on CRN Proteins in the Genomics Age: Evolution, Classification, Delivery and Function Revisited.

Plant associated microbes rely on secreted virulence factors (effectors) to modulate host immunity and ensure progressive infection. Amongst the secreted protein repertoires defined and studied in pathogens to date, the CRNs (for CRinkling and Necrosis) have emerged as one of only a few highly conserved protein families, spread across several kingdoms. CRN proteins were first identified in plant pathogenic oomycetes where they were found to be modular factors that are secreted and translocated inside host cells by means of a conserved N-terminal domain. Subsequent localization and functional studies have led to the view that CRN C-termini execute their presumed effector function in the host nucleus, targeting processes required for immunity. These findings have led to great interest in this large protein family and driven the identification of additional CRN-like proteins in other organisms. The identification of CRN proteins and subsequent functional studies have markedly increased the number of candidate CRN protein sequences, expanded the range of phenotypes tentatively associated with function and revealed some of their molecular functions toward virulence. The increased number of characterized CRNs also has presented a set of challenges that may impede significant progress in the future. Here, we summarize our current understanding of the CRNs and re-assess some basic assumptions regarding this protein family. We will discuss the latest findings on CRN biology and highlight exciting new hypotheses that have emanated from the field. Finally, we will discuss new approaches to study CRN functions that would lead to a better understanding of CRN effector biology as well as the processes that lead to host susceptibility and immunity.

Utilizing "Omic" Technologies to Identify and Prioritize Novel Sources of Resistance to the Oomycete Pathogen Phytophthora infestans in Potato Germplasm Collections.

The greatest threat to potato production world-wide is late blight, caused by the oomycete pathogen Phytophthora infestans. A screen of 126 wild diploid Solanum accessions from the Commonwealth Potato Collection (CPC) with P. infestans isolates belonging to the genotype 13-A2 identified resistances in the species S. bulbocastanum, S. capsicibaccatum, S. microdontum, S. mochiquense, S. okadae, S. pinnatisectum, S. polyadenium, S. tarijense, and S. verrucosum. Effector-omics, allele mining, and diagnostic RenSeq (dRenSeq) were utilized to investigate the nature of resistances in S. okadae accessions. dRenSeq in resistant S. okadae accessions 7129, 7625, 3762, and a bulk of 20 resistant progeny confirmed the presence of full-length Rpi-vnt1.1 under stringent mapping conditions and corroborated allele mining results in the accessions 7129 and 7625 as well as Avr-vnt1 recognition in transient expression assays. In contrast, susceptible S. okadae accession 3761 and a bulk of 20 susceptible progeny lacked sequence homology in the 5' end compared to the functional Rpi-vnt1.1 gene. Further evaluation of S. okadae accessions with P. infestans isolates that have a broad spectrum of virulence demonstrated that, although S. okadae accessions 7129, 7625, and 7629 contain functional Rpi-vnt1.1, they also carry a novel resistance gene. We provide evidence that existing germplasm collections are important sources of novel resistances and that "omic" technologies such as dRenSeq-based genomics and effector-omics are efficacious tools to rapidly explore the diversity within these collections.