Phytophthora infestans avirulence genes: mapping, cloning and diversity in field isolates

Potato late blight, caused by the oomycete pathogen Phytophthora infestans (Mont.) de Bary, is the most disastrous disease on potato worldwide and also the greatest threat to potato production in China. Loss of yield and quality, and the costs of chemical control of potato late blight account for multi-billion US$ annually. Using host resistance is thought to be an economical and efficient approach to control potato late blight. To combat the disease breeders have introduced late blight resistance (R) genes from various Solanum species into the cultivated potato. The proteins encoded by these R genes can recognise specific races of the pathogen. This then triggers a cascade of defence responses ultimately leading to a localized programmed cell death called the hypersensitive response (HR) that arrests growth of the pathogen. However, once the resistant potato cultivars are released into the field, the resistance based on these genes is quickly overcome due to rapid evolution and adaption of P. infestans. <br/> In the potato-P. infestans interaction race-specific recognition by R genes is based on the ‘gene-for-gene’ model which predicts that resistance is governed by the (direct or indirect) interaction of an R protein with its corresponding effector, the product of an avirulence (Avr) gene. If either the R gene or the Avr gene is absent or non-functional the interaction is compatible and the host susceptible for disease. Key to a better understanding of the molecular basis of resistance in the potato-P. infestans pathosystem is the unravelling of R protein-effector interactions and, hence, cloning of more R and Avr genes is a prerequisite to study these interactions. This thesis describes the mapping and cloning of Avr genes in P. infestans, and the phenotypic and genotypic diversity in P. infestans field isolates in Northern China. <br/> To isolate P. infestans Avr genes a positional cloning strategy was adopted. Chapter 2 presents a molecular-genetic linkage map of P. infestans that was constructed based on Single Nucleotide Polymorphism (SNP) markers and Amplified Fragment Length Polymorphism (AFLP) markers. The map was generated using a mapping population of 83 F1 progeny derived from two Dutch field isolates, NL80029 and NL88133. Of 631 markers (398 SNP and 233 AFLP markers) that segregated in this population, 534 markers were positioned on 19 linkage groups spanning a total of 1144 cM and an average distance of 2.14 cM between adjacent markers. Fourteen of the linkage groups are major linkage groups that contain markers from both parents. The others are minor linkage groups with markers of only one of the two parents. <br/> In parallel, a transcriptional profiling strategy was adopted to identify avirulence-associated transcripts (Chapter 3). cDNA-AFLP was used for comparing transcripts in P. infestans isolates with different virulence phenotypes. A large number of avirulence-associated TDFs (Transcript Derived Fragments) was cloned and sequenced, and EST and genome databases were mined to generate more sequence data. To identify promising candidates, bioinformatic predictions such as the presence of signal peptides, number of cysteine residues and putative virulence functions were used as important selection criteria. Four TDFs associated with Avr loci were identified, two for Avr4 and two for the Avr3b-Avr10-Avr11 locus. <br/> Chapter 4 describes how a combined approach of genetic mapping, transcriptional profiling and BAC marker landing resulted in isolation of the P. infestans avirulence gene Avr4. PiAvr4 encodes a 287 amino acid protein that belongs to a superfamily of effectors sharing the putative host cell targeting motif RXLR-dEER. For the functional characterization P. infestans race 4 strains were transformed with PiAvr4. This resulted in transformants that were avirulent on R4 potato plants, demonstrating that PiAvr4 is responsible for eliciting R4-mediated resistance. Expression of PiAvr4 in R4 plants using PVX-agroinfection and agroinfiltration showed that PiAvr4 itself is the effector that elicits HR on R4 plants. On potato plants lacking R4, like Bintje, there was no response. The presence of the RXLR-dEER motif suggested intracellular recognition of PiAvr4 but nevertheless a hypersensitive response was observed when PiAvr4 was targeted to the outside of the cell. Deletion of the RXLR-dEER domain neither stimulated nor prevented elicitor activity of PiAvr4. Race 4 strains have frame shift mutations in the PiAvr4 gene that result in short truncated peptides, indicating that PiAvr4 is not crucial for virulence. <br/> Chapter 5 describes Avr1-associated markers that resulted from genetic mapping, transcriptional profiling and BAC-end sequences. In silico landing of these markers on the P. infestans genome sequence narrows down a 800 kb genomic interval that carries seven genes that have the hallmarks of an oomycete Avr gene. They all encode a secreted protein with a conserved RXLR-dEER domain at the N-terminus and a divergent C-terminal region. Each of these seven could be a candidate for Avr1. The seven RXLR effector genes were further characterized by bioinformatic analyses such as HMM score of the RXLR motif, and prediction of the presence of W, Y, and L motifs in the C-terminal region. Cloning and functional analyses using transient expression assays in plants carrying the resistance gene R1 should reveal whether any of the seven candidates is Avr1. <br/> Chapter 6 describes the phenotypic and genotypic diversity of P. infestans isolates collected in Northern China between 1997 and 2003, especially in Inner Mongolia. Characterization included mating type, virulence, mitochondrial DNA (mtDNA) haplotype and DNA fingerprinting patterns based on simple sequence repeats (SSR) and amplified fragment length polymorphism (AFLP). All isolates had the A1 mating type, mtDNA haplotype IIa and an identical SSR genotype (designated as SG-01-01) that differed from the SSR genotypes found in the reference isolates, including the ones representing the ‘old’ US-1 lineage that dominated the worldwide P. infestans population prior to 1980. In contrast, the virulence spectra differed significantly and virulence to all R genes present in the standard differential set (R1 to R11) was found. AFLP analysis revealed some diversity; eight different AFLP genotypes were found that could be grouped into two major clusters. This study shows that there is very little genotypic diversity in the P. infestans population in Northern China. The occurrence of many different races within this uniform population is discussed in the framework of recently gained insights in the molecular determinants of avirulence in P. infestans and their role in the ‘gene-for-gene’ interaction with potato. <br/> Finally, in Chapter 7, the implications of the findings described in this thesis are discussed with specific emphasis on Avr gene cloning, RXLR-dEER effectors, virulence diversity and durable late blight resistance. By combining various cloning strategies it becomes feasible to speed up the cloning of putative P. infestans Avr genes. Moreover, the use of high throughput effector genomics screenings will allow the identification of the corresponding R genes. The high virulence diversity that is found in P. infestans field isolates, even within one clonal lineage, might be correlated to the observation that RXLR-dEER effector genes are the most rapidly evolving genes in the genome of P. infestans. Therefore, generating potato cultivars with durable resistance to late blight seems more challenging than anticipated. <br/>