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Description
A main objective of the project is to improve our understanding of interaction and co-evolution of host and pathogen using potato (Solanum tuberosum L.) and its economically most important pathogen, Phytophthora infestans (Mont.) de Bary, as a model. Our main hypothesis is that co-evolution of potato and P. infestans in agroecosystems is based on diversity of the resistance genes present in the cultivated potatoes and effector genes present in the pathogen populations. DivGene project objectives include analyses of the diversity of the potato R genes conveying resistance against P. infestans and of the effector genes of P. infestans. The availability of new technology, combined with large collections of P. infestans isolates and potato cultivars, make the proposed research both novel and feasible. For P. infestans, the project partners IHAR-PIB in Poland and NIBIO in Norway have around 1000 isolates each, characterized both phenotypically and genetically using SSR markers. IHAR-PIB has a collection of over 1600 potato cultivars stored by National Centre for Plant Genetic Resources: Polish Genebank, and NIBIO has an in vitro collection of all available cultivars in Norway. The novelty and originality of our approach is lying in the fact that we propose the diversity analyses of key genes involved in the potato – P. infestans interaction on a population scale, expanding both the numbers of analyzed genes and objects (plant and pathogen genotypes). The results of the AmpSeq-based diversity studies will be complemented with phytopathological tests in which host resistance and pathogen virulence and aggressiveness will be verified and linked to the sequence diversity of the studied genes. We will also perform an expression study of the chosen host and pathogen genes at selected time points during the interaction. Populations of host and pathogen will be studied in diverse conditions of Poland and Norway, thus enforcing our findings.
Summary of project results
A main objective of the DivGene project was to improve our understanding of interaction and co-evolution of host and pathogen using potato (Solanum tuberosum L.) and its economically most important pathogen, Phytophthora infestans (Mont.) de Bary, as a model. Potato late blight caused by P. infestans is currently controlled in potato production by intensive application of pesticides. Using host resistance would be a desirable alternative. Almost 6000 potato cultivars, historical and currently in use, are listed in the European Cultivated Potato Database, and 1882 of them are described as medium to very highly resistant to late blight. However, in the vast majority of the cultivars we do not know what genes underlie their resistance. The breeders often do not know what resistance (R) genes are present in their materials, or they do not reveal the sources of intentionally introduced resistance. The potato cultivar pedigrees, if available, may also be misleading and incorrect. The picture is additionally complicated by the fact that many R genes still existing in the cultivars turned ineffective due to the fast evolution of pathogen races able to overcome the resistance they provide.
From the pathogen side, only 10 effectors that encode proteins recognized by known R proteins of potato are known, out of 550 effector genes in the sequenced genome of P. infestans. How they manipulate the host defense or cell structure is only known for a few of them. The diversity of effectors is potentially very high, which is crucial for the interactions with host plants. However, diversity studies so far have been limited, in very few isolates of the pathogen.
The DivGene project aimed to apply next generation sequencing technologies to unravel presence and diversity of potato late blight resistance genes in potato cultivars grown in Poland and Norway, as well as the diversity of P. infestans effectors in a representative set of isolates collected in Poland and Norway.
a. The Amplicon Sequencing (AmpSeq) approach was tested the Rysto gene derived from the wild potato relative Solanum stoloniferum that confers extreme resistance to PVY and the Rpi-vnt1 (Rpi-phu1) gene from S. phureja that confers resistance to P. infestans. The presence and diversity of the Rysto gene was investigated in wild relatives of potato (298 genotypes representing 29 accessions of 26 tuber-bearing Solanum species). In a single PacBio sequence run, we obtained 8.35 Gb from 99 amplicons, of which 91 amplicons were Rysto homologues of ca. 5 kb and eight were Rpi-vnt1 homologues of ca. 2 kb.
b. Diversity of 10 selected potato resistance genes was analysed by AmpSeq in a set of 335 potato genotypes, including 183 potato cultivars, 98 breeding lines and 54 wild potato species. The potato genotypes were selected from traditional and contemporary potato cultivars in Poland and Norway, as well as new cultivars and breeding lines. A mixture of genotypes with varying levels of resistance to late blight was included: from resistant to susceptible ones. As a result, 6 million raw reads were obtained, which yielded 4 million HiFi reads of average subread length 4088 bp.
c. Diversity of more than 60 selected P. infestans genes was analysed by AmpSeq in a set of 394 isolates from Poland and Norway. We found considerable effector variation between isolates that were previously classified as the same genotype through length polymorphisms in twelve simple sequence repeat markers (SSRs), which is the traditional method for characterizing genotypes. Effector variation included SNPs leading to amino acid substitution and premature stop codons. One to eleven variants were observed per gene with a median of three.
d. Phenotypes of selected potato and P. infestans genotypes were evaluated and gene expression in the host and the pathogen were measured in laboratory infection assays.
e. Full genomes of two P. infestans isolates from Poland (MP1831; MP324x) and three from Norway (N16-009; N16-105; N17-176) were sequenced using PacBio high fidelity (HiFi) long read sequencing. A total of 86.47 Gb HiFi reads with an average read length of 17 kilobases (kb) was obtained. The reads were de novo assembled to 242 to 287 megabases genomes in 2,511 to 4,052 contigs, with N50 values ranging from 142,353 to 254,961 bp and coverage from 49x to 69x.
Divgene project outcomes and impacts:
- For scientific community: new knowledge on P. infestans and potato interactions, Rysto gene diversity (scientific publications), new DNA sequence resources (raw reads available in NCBI Sequence Read Archive (SRA), potato and P. infestans gene sequences available as the NCBI GenBank accessions, new publicly available P. infestans genome assemblies in the DDBJ/ENA/GenBank repository), new bioinformatic tools (code used to process the raw data with Lima and DADA2 and intermediary files can be found in the GitLab repository).
- For potato breeders: knowledge on distribution and diversity of Rpi genes in the breeding lines and potato cultivars, sequence data useful for marker-assisted selection (reports delivered to the three collaborating breeding companies), knowledge on P. infestans effectors’ diversity and effectiveness of potato resistance genes useful for planning thee breeding strategies.
- For potato growers and agricultural advisors: new knowledge on late blight resistance in potato cultivars, promoting host resistance as a sustainable measure for control of the disease, contributing to reduction of the pesticide use in potato production.
Summary of bilateral results
The most important advantage of the international cooperation was that it allowed to test the research hypothesis in two different agroecosystems of Poland and Norway. That broght stronger evidence, better quality of data and more justified conclusions that were published in scientific journals with high impact. Most of the project tasks were performed in bilateral collaboration between Polish and Norwegian teams. Contribution of both teams is confirmed with joint authorship of the published/submitted research paper/manuscript.