Molecular tools to unravel the role of genes from Phytophthora infestans

The oomycete plant pathogen Phytophthora infestans is the causal agent of potato late blight. P. infestans is undoubtedly the best known and most studied Phytophthora species today. This is mainly because it is such a devastating pathogen that can cause complete destruction of a potato field in only a few days. In this thesis, we describe the characterisation of four P. infestans genes with presumed functions in pathogenicity and virulence, and the development of tools to study expression and function of two of these genes in P. infestans . The four genes are the in planta -induced genes ipiB and ipiO , the elicitin gene inf1 , and the stress-induced gene ric1 . We used theβ-glucuronidase reporter gene for expression analysis of the ipiO gene, and homology-dependent gene silencing for functional analysis of the inf1 gene. The latter resulted in the discovery of a new phenomenon, which we named internuclear gene silencing.In Chapter 2, we describe the characterisation of the in planta -induced genes ipiB and ipiO . IpiB constitutes a gene family with at least three members, ipiB1 , ipiB2 and ipiB3 , which are clustered in a head-to-tail arrangement. The ipiB genes are highly homologous throughout their promoter, coding and terminator sequences and encode three similar glycine-rich proteins of 301, 343 and 347 amino acids, respectively. The glycine-rich domains of the IPI-B proteins are predominantly composed of two repeats with the core sequences, A/V-G-A-G-L-Y-G-R and G-A-G-Y/V-G-G. The IPI-B proteins contain a putative signal peptide of 20 amino-acids, suggesting that the proteins are targeted to a specific organelle or to the outside of the cell. We speculate that the IPI-B proteins are structural proteins associated with the cell wall that are possibly involved in the development of infection structures. Also ipiO is a small gene family. Two members, ipiO1 and ipiO2 are closely linked and arranged in an inverted orientation. The ipiO genes encode almost identical 152 amino acid-proteins which do not have any significant homology with sequences present in data libraries. The IPI-O proteins contain a putative signal peptide which may target them to the extracellular matrix. A putative cell attachment sequence (RGD), functional in mammalian systems, was identified.Expression of the ipiO gene was analysed during several developmental stages of the life cycle of P. infestans (Chapter 3). IpiO mRNA was detected in zoospores, cysts, and germinating cysts, but not in sporangia or in mycelia grown in vitro . IpiO is also expressed during colonisation of potato leaves. In disease lesions, ipiO mRNA was detected in the water-soaked area and the surrounding healthy-looking plant tissue. IpiO mRNA could not be detected in necrotised tissue and sporulating areas of a lesion. Cytological assays were performed to determine more precisely the location and time of ipiO gene expression in planta . P. infestans transformants expressing a transcriptional fusion between the ipiO1 promoter and theβ-glucuronidase (GUS) reporter gene showed that GUS staining was specifically found in the subapical and vacuolated area of tips of invading hyphae. Therefore, we concluded that ipiO is expressed in hyphae during biotrophic stages of the infection process. We speculate that IPI-O has its function in the hyphal tips at the edge of the expanding lesion where the pathogen is invading healthy leaf cells. The IPI-O protein may well be localised at the interface between the invading hyphae and the plant cells, and could play a role in pathogenicity and/or virulence.To isolate more genes that might be involved in the interaction between P. infestans and potato, we used a small scale expressed sequence tag (EST) approach (Chapter 7). Twenty-two P. infestans cDNA clones were randomly selected from a potato- P. infestans interaction cDNA library. Five of these clones appeared to be derived from the same gene, ric1 . Two copies of the ric1 gene were identified and both genes share 98% homology at the nucleotide sequence level and 100% at the amino acid level. The open reading frame predicts a small protein sequence of 57 amino acids. The highly hydrophobic protein has two potential membrane spanning domains. The deduced amino acid sequence shows high homology with three putative plant proteins encoded by genes of which the expression is specifically induced during stress conditions. Expression of ric1 increased considerably during osmotic stress and at high pH. <strong></strong> We hypothesise that RIC1 is a structural protein that is necessary to maintain membrane integrity, especially during unfavourable conditions.When culturing various Phytophthora species in liquid medium, all examined species secrete highly abundant 10 kDa proteins which have been shown to elicit a hypersensitive response when injected into tobacco leaves. It was hypothesised previously that these 10 kDa proteins, collectively called elicitins, function as plant species specific avirulence factors. In order to test this hypothesis, we set out to clone the elicitin gene inf1 , of P. infestans (Chapter 4) and subsequently generated mutants that no longer produced the INF1 protein (Chapter 5 & 6). An inf1 cDNA was isolated by heterologous hybridisation of a potato- P. infestans interaction cDNA library using parA1 , the gene encoding the major secreted elicitin of P. parasitica , as a probe. Inf1 encodes a 118 amino acid protein including a 20 amino acid signal peptide. Detailed expression studies show that inf1 is expressed in mycelium grown in various culture media, whereas expression was not detected in sporangia, zoospores, cysts and germinating cysts. The highest levels of expression of inf1 are observed in in vitro -grown mycelium and in planta during the late stages of infection when profuse sporulation and leaf necrosis occur. Expression is, however, down-regulated during early biotrophic stages of the interaction.To obtain P. infestans strains deficient in INF1 production, we explored a homology-dependent gene silencing strategy. Integrative transformation with antisense, sense and promoter-less constructs in P. infestans was performed to generate mutants. Inf1 mRNA and INF1 protein could not be detected in up to 20% of the transformants. The silenced state of the inf1 gene was shown to be mitotically stable under various conditions in vitro and in planta . In pathogenicity assays, the INF1-deficient strains remained pathogenic on the host plants potato and tomato. However, in contrast to the wild type and control transformant strains, INF1-deficient strains induced also disease lesions and extensive sporulation when inoculated on Nicotiana benthamiana . These results demonstrate that recognition of INF1 elicitin leads to active resistance in N. benthamiana to P. infestans and that INF1 functions as a plant species-specific avirulence factor.In Chapter 5, we describe experiments to identify the mechanism of silencing of the inf1 gene in P. infestans . Nuclear run-on assays showed that inf1 gene silencing is regulated at the transcriptional level. Interestingly, DNA methylation, a feature often associated with transcriptionally regulated gene silencing could not be detected in the inf1 gene sequences. Heterokaryons obtained by somatic fusion of an inf1 -silenced transgenic strain and a wild type strain displayed also stable gene silencing, demonstrating that inf1 silencing is dominant and acts in trans. The inf1 gene remained silenced in non-transgenic homokaryotic single zoospore isolates released from the silenced heterokaryons. Apparently, the presence of transgenes is not essential for maintaining the silenced status of the endogenous inf1 gene. Karyogamy was not demonstrated and hence, it is unlikely that the silenced state of the inf1 gene is transmitted from nucleus to nucleus by specific DNA-DNA interactions. Consequently, we propose a novel silencing phenomenon, called internuclear gene silencing, in which a diffusible silencing factor is involved in inducing stable gene silencing. We speculate that the proposed diffusible trans -acting silencing factor is either a protein, an aberrant RNA molecule or a complex consisting of RNA and protein. We envisage that such a molecule is transported from nucleus to nucleus where it may facilitate an inheritable change in inf1 -expression possibly initiated by changes in chromatin structure of the target gene or regions surrounding the target gene. Internuclear gene silencing is clearly a trans -inactivation phenomenon that is reminiscent of paramutation.In Chapter 8, we discuss how the results presented in this thesis will contribute to a better understanding of the biology and pathogenicity of P. infestans . On the long run, increased knowledge of this notorious pathogen will help in developing alternative methods to control potato late blight