The Phytophthora infestans avirulence gene PiaAvr4 and its potato counterpart R4

The potato late blight disease that is caused by the oomycete pathogen Phytophthora infestans is a major threat for potato crops worldwide. In recent years research on oomycete plant pathogens was boosted by the availability of novel genomic tools and resources for several oomycete genera, such as Phytophthora, Hyaloperonospora, Pythium and Aphanomyces. This has led to the identification of genes involved in diverse biological processes such as sporulation, mating, signaling and pathogenesis. One of the approaches that breeders use to obtain late blight resistant potato cultivars is the introgression of resistance traits from wild Solanum species into the cultivated potato Solanum tuberosum. The pathogen, however, is able to circumvent this resistance; it is often lost shortly after introduction of new cultivars. To better understand the mechanisms underlying this loss of resistance it is of utmost importance to gain insight into the characteristics of the cognate avirulence (Avr) genes of the pathogen. According to the gene-for-gene model Avr genes encode effectors that trigger resistance responses mediated by resistance (R) genes. This thesis first describes the identification of a P. infestans Avr gene, in particular the elicitor activity of the encoded effector protein, the domain structure of the effector and its putative sub-cellular localization. In the second part the recognition specificity of the corresponding R gene and the identification of a marker linked to this R gene are described. Chapter 1 summarizes the advances in oomycete genomics in recent years and the tremendous progress that has been made in gene discovery in oomycete plant pathogens. It describes the different oomycete species that have been studied in more detail and assesses which species are suitable model species for research on oomycete-plant interactions. The identification of the P. infestans avirulence gene PiAvr4 is presented in Chapter 2. PiAvr4, which encodes an RXLR-dEER effector protein, was isolated by positional cloning. AFLP markers were used for landing on BACs and cDNA-AFLP markers pinpointed the gene of interest. Transformation of race 4 strains with PiAvr4 resulted in transformants that are avirulent on the R4 differential of the Mastenbroek differential set (clone Ma-R4). Moreover, in planta expression of PiAvr4 resulted in a necrotic response on clone Ma-R4 but not on plants lacking R4 such as Bintje. All together this proves that PiAvr4 is the avirulence gene that corresponds to the R gene present in clone Ma-R4. In many identified avirulence proteins one or a few amino acid changes in the protein abolish avirulence function. In case of PiAvr4, race 4 strains have frame shift mutations in the open reading frame, resulting in a truncated protein that is not functional as avirulence factor. Effectors within the RXLR-dEER family are rapidly evolving. The selective pressure is targeted predominantly on the C-terminal region of these proteins. Despite this selective pressure the majority of these proteins carry motifs that can be distinguished using Hidden Markov Models searches. They are named W, Y and L motifs after the conserved tryptophan (W), tyrosine (Y) and leucine (L) residues, respectively. As described in Chapter 3 PiAvr4 carries three W motifs and a single Y motif. The motifs together with their flanking regions were tested for activity on Ma-R4 plants. Agroinfection of constructs carrying the W2 motif in combination with either the W1 or W3 motif resulted in a necrotic response. Moreover, we showed that the PiAvr4 homolog PmirAvh4, isolated from Phytophthora mirabilis was also able to elicit a necrotic response on the Ma-R4 potato clone. For several Phytophthora RXLR-dEER effectors it was demonstrated that these proteins are targeted into the host cell and that the RXLR-dEER motif is required for translocation. In Chapter 4 we investigated whether PiAvr4 and IPI-O, like other RXLR-dEER effectors, are also targeted into the host cell. A race 4 P. infestans isolate was transformed with constructs encoding either PiAvr4 or IPI-O fused to a monomeric red fluorescent protein (mRFP) at the C-terminus. Fluorescence microscopy of these transformants showed no specific mRFP fluorescence in free living, non-sporulating mycelium. However, in germinating cysts, the tips of germ tubes and appressoria showed mRFP fluorescence, and during infection of etiolated potato plantlets localized fluorescence was visible at the haustorial neck. Haustoria are highly specialized infection and feeding structures that are in close contact with the plant cell and have a putative role in delivering effector proteins into the host cell. In order to monitor the development of the infection a novel experimental set-up was developed. In this method etiolated in vitro grown potato plantlets are inoculated with P. infestans, which has the advantage that there is no autofluorescence of chlorophyll that masks the mRFP fluorescence and thus disturbs the microscopic analysis in green plant tissues. The lack of chlorophyll does not seem to interfere with infection; zoospores are capable to encyst and to germinate, and the etiolated tissues are readily colonized by P. infestans. The recognition specificity of R4 potato differentials is described in Chapter 5. Initially two different potato clones were developed as R4 differentials; The Mastenbroek differential set, developed in the Netherlands, contains the clone Cebeco44-31-5 (designated as Ma-R4) and the Black differential set, developed in Scotland, contains clone 1563 c (14) (designated as Bl-R4). Virulence assays using several wild type P. infestans strains revealed that the Bl-R4 clone is susceptible to all isolates that are avirulent on clone Ma-R4. Only one single isolate was found to be avirulent on clone Bl-R4, but virulent on Ma-R4. Moreover, in transient expression assays with binary PVX constructs carrying PiAvr4, the Ma-R4 clone but not the Bl-R4 clone responded with an HR. Similar to the R3 locus two different recognition specificities seem to exist for R4. The R3a and R3b genes are located on one locus but whether this is the case for the two R4 genes (named R4Ma and R4Bl, respectively) remains to be determined. Resistance to P. infestans strains carrying PiAvr4 segregates in an 1:1 ratio in two independent potato F1 populations suggesting that R4Ma resistance is determined by a single dominant locus. More in depth studies on the recognition of PiAvr4 by its cognate R protein are hampered by the fact that the resistance gene R4Ma has not yet been identified. In Chapter 6 nucleotide binding site (NBS) profiling was used to generate R4Ma-associated markers. NBS profiling is a biased approach based on PCR amplification of conserved NBS motifs in R genes and R gene homologs. In a bulked segregant analysis, DNA of resistant and susceptible F1 progeny was pooled and used as template for NBS profiling. Several candidate markers were found but eventually one amplified fragment was found to co-segregate with resistance mediated by R4Ma. DNA sequencing of this fragment revealed high similarity to BAC sequences that are mapped to potato chromosome 12. Moreover, the R4Ma marker is homologous to members of the Rx/Gpa2 gene family. Chapter 7 focuses on the secreted effectors of plant pathogenic oomycetes, with special attention to RXLR-dEER effectors, and the role of these proteins in pathogenesis. The RXLR-dEER effector family is rapidly evolving and comprises all secreted oomycete avirulence proteins that are identified up till now. There is now ample evidence that oomycetes utilize the RXLR-dEER domain to deposit effectors inside host cells. Furthermore, this chapter discusses the experimental results described in this thesis in the light of present knowledge on gene-for-gene interactions, effector recognition and late blight resistance.