Differential gene expression in Phytophthora infestans during pathogenesis on potato = [Differentiele genexpressie in Phytophthora infestans tijdens de pathogenese op aardappel]

Abstract

The plant pathogenic fungus Phytophthora infestans (Mont.) de Bary is the causal agent of potato late blight, the most important and devastating fungal disease of potato. The molecular and cellular processes involved in pathogenicity of this hemibiotrophic fungus are poorly understood. The aim of the research described in this thesis was to gain more insight in these processes. A novel and unbiased approach was explored to isolate P . infestans genes which might have a function in pathogenesis. The approach was based on the assumption that the expression of pathogenicity genes of P . infestans , i.e. genes which are essential for the establishment and maintenance of basic compatibility or for the increase of disease severity, is specifically induced or significantly increased during pathogenesis. The strategy implies the isolation of in planta induced ( ipi ) genes by differential screening of a genomic library of P.infestans , subsequent characterization of their gene products, and elucidation of their function in pathogenesis.By differential screening, nine distinct in planta induced genes were isolated, i.e. ipiA , ipiB , ipiC , ipiD , ipiJ1 , ipiJ2 , ipiN , ipiO and ipiQ . Expression studies revealed that the ipiB and ipiO genes have a transient expression pattern during pathogenesis with the highest levels in early stages of the interaction. The seven other ip i-genes show a continuous 5-10 fold increase in expression level during growth of the fungus in the host.The P.infestans genes ipiA , ipiB , ipiC and ipiO were further characterized. Two of these appeared to encode ubiquitous proteins: ipiA , which was renamed ubi3R , encodes ubiquitin and ipiC , renamed calA , codes for calmodulin. Both proteins are highly conserved in eukaryotic organisms and have been shown to play important roles in basic cellular processes such as selective degradation of abnormal proteins (ubiquitin) and signal transduction (calmodulin). This is the first time that an increased expression of ubiquitin and calmodulin genes is found in association with pathogenicity of a plant pathogenic fungus. However, the significance of increased ubiquitin and calmodulin levels in P.infestans during pathogenesis remains to be resolved. The ipiB gene belongs to a gene family consisting of at least three highly homologous genes, ipiB1 , ipiB2 and ipiB3 , which are clustered on the genome in a head-to-tail arrangement. The ipiB genes encode novel glycine-rich proteins (GRPs) of 301 (IPI-B1), 343 (IPI-B2), and 347 (IPI-B3) amino acids (aa) respectively. The primary structure of the IPI-B proteins is composed of a N-terminal signal sequence followed by a large, highly repetitive glycine-rich domain. The glycine-rich domain is 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, respectively. The characteristic primary structure is also found in plant GRPs of which some have been shown to be associated with the plant cell wall. The P. infestans genome contains two ipiO genes, ipiO1 and ipiO2 , which are closely linked and arranged in an inverted orientation. The ipiO genes encode two nearly identical 152 aa proteins which have no homology with any known sequence. As the IPI-B proteins, the IPI-O proteins contain a N-terminal signal sequence suggesting that they are excreted. In addition, the IPI-O proteins have a putative N-glycosylation site and a typical Arg-Gly-Asp (RGD) tripeptide motif. The RGD tripeptide is conserved in several mammalian extracellular adhesion proteins in which it functions as a cell attachment motif. Whether the IPI-O proteins have adhesion properties is unknown.The four in planta induced P . infestans genes ubi3R , calA , ipiB and ipiO , are among the first of the limited group of oomycetous genes to be isolated and sequenced. As in most known oomycetous genes, the coding sequence of these genes is not interrupted by introns. Within 100 nucleotides upstream of their ATG start codon, a conserved motif matching the consensus sequence GCTCATTYYNCA(A/T)TTT was identified. Comparison of the 5' nontranscribed regions of eight distinct oomycetous genes revealed that this sequence motif surrounds the transcription start point of the majority of these genes, suggesting that oomycetous genes have a GCTCATTYYNCA(A/T)TTT sequence preference for transcription initiation.The expression of the ipiB and ipiO genes was studied in more detail. The ipiB genes as well as the ipiO genes are expressed in germinating cysts prior to host penetration. During pathogenesis both genes are transiently expressed. The highest mRNA levels are found in early stages of infection. Also during initial stages of interactions of P.infestans with a racespecific resistant host and the nonhost Solanum nigrum L., the expression of the ipiB and ipiO genes is induced. During growth of the fungus in vitro, nutrient deprivation appeared to be a strong stimulus for the induction of ipiB and ipiO gene expression. For several other pathogenesis related genes characterized in fungal plant pathogens it has been shown that nutrient starvation induces their expression. Whether starvation conditions mimic the nutrient condition encountered upon infection and, as such, function as a general stimulus for the activation of pathogenicity genes remains to be established.Manipulation of gene expression in P.infestans is an important tool to study gene function. The possibility to manipulate gene expression in P. infestans by anti-sense RNA was tested. First, the promoters of five oomycetous genes, among which the P.infestans ubi3R gene, were fused to the reporter β-glucuronidase ( GUS ) gene of E. coli and the activity of the promoters was determined in transient expression assays as well as in stable transformants. The hsp70 and ham34 genes of Bremia lactucae appeared to have the highest activity. When the GUS gene in the anti-sense orientation fused to the ham34 promoter, is introduced in transgenic P.infestans containing sense GUS constructs, the accumulation of GUS is effectively inhibited. This indicates that the anti-sense technique is suitable to study gene function in P.infestans.</em