Functional analysis of tomato genes expressed during the Cf-4/Avr4-induced hypersensitive response

Plant diseases caused by pathogenic micro-organisms can result in severe crop losses and are a threat for global food and feed production and quality. Resistance breeding based on the introduction of a single dominant resistance gene is often not durable, as under selection pressure pathogens are able to overcome resistance by circumventing recognition by the host. After perception of a pathogen by a plant many different defense signaling pathways are activated, which are likely more difficult to avoid by the pathogen than the initial recognition event. Furthermore, these downstream responses are probably conserved among different plant species and are effective against different pathogens. The goal of the present study is to employ the tomato- Cladosporium fulvum interaction as a model to identify genes involved in defense signaling pathways. Identification of these genes increases our understanding of cellular responses of plants resistant to pathogens. This knowledge should eventually be exploited to develop durable resistant plants, either by marker-assisted breeding or by transgenesis.

C. fulvumis a foliar, biotrophic fungal pathogen which causes leaf mold disease of tomato. In resistant tomato plants, within one to two days post penetration a microscopic H ypersensitive R esponse (HR) is observed preventing further growth of the biotrophic fungus. Tomato is the only host for C. fulvum and recognition of specific avirulence (Avr) factors encoded by Avr genes of the fungus is mediated by matching resistance proteins encoded by Cf genes of the plant, following the gene-for-gene model. Besides this host-specific and gene-for-gene-based resistance, mechanisms providing passive- and active- non-host resistance and some of the downstream defense responses are presented in a short overview (chapter 1).

To identify genes required for Cf/Avr- induced defense pathways, we used a system that allows synchronized induction of defense responses by one Cf/Avr gene pair without the interference of additional fungal genes or proteins, referred to as the "dying seedling system". Therefore, a near-isogenic tomato line carrying Cf-4 was crossed to tomato transgenic for the Avr4 gene (and lacking functional Cf genes), resulting in offspring carrying the Cf-4/Avr4 gene pair. When such plants are grown at 33ºC the HR is suppressed. Subsequent shifting these plants to room temperature results in a synchronized HR. RNA was isolated from Cf-4 controls and Cf-4/Avr4 seedlings at 0, 30, 60 and 90 minutes after the temperature shift and subsequently cDNA-AFLP analysis was performed. This resulted in 442 differentially expressed cDNA-AFLP fragments corresponding to genes putatively involved in plant defense, designated A vr4- R esponsive T omato ( ART ) genes. To determine the requirement of the corresponding genes for plant defense V irus- I nduced G ene S ilencing (VIGS), a technique that allows generating fast transient individual "knock-downs" of relatively large sets of genes, was used. We first generated knock-downs for a set of 192 selected ART fragments in Nicotiana benthamiana transgenic for Cf-4 . VIGS of ART genes encoding a heat shock protein 90, a nuclear GTPase, an L19 ribosomal protein and, most interestingly, an NB-LRR protein clearly affected both Cf-4/Avr4- and Inf1- induced HR. The latter is an elicitor of Phytophthora infestans on its non-host N. benthamiana . Since VIGS using the ART fragment encoding the NB-LRR protein affected both the Cf-4/Avr4 and Inf1- induced HR in N. benthamiana, the protein was designated NRC1 ( N B-LRR R equired for HR-associated C ell death 1) (chapter 2) .

Chapter 3describes the high-throughput functional analysis using VIGS in tomato to score for genes required for resistance to C. fulvum. VIGS was optimized in tomato using a fragment of the phytoene desaturase ( PDS ) gene. Successful silencing of PDS is visible as white tissue, thus providing a visual marker for silencing. Subsequently, VIGS of the resistance gene Cf-4 and a gene required for Cf-2- mediated resistance, Rcr3 was performed and in addition, a method to sensitively detect growth of C. fulvum in tomato leaves was optimized . Finally, we performed VIGS using the 192 selected ART fragments followed by inoculation with the fungus. VIGS using four of the selected 192 ART fragments, encoding a histon H3 protein, a ribosomal protein L1, an unknown protein and the NB-LRR protein, resulted in decreased resistance of tomato to C. fulvum.

The research described in chapter 4 is focused on the NRC1 gene , which is the first NB-LRR encoding gene required for Cf- mediated defense. Although NRC1 proved to be required for multiple HR pathways, silencing of the gene only affected resistance to C. fulvum . Transient expression of a constitutively active mutant of NRC1 resulted in an elicitor-independent HR. Epistasis experiments revealed that the NRC1-induced HR is dependent on MEK2, SGT1 and RAR1, but independent of NDR1. It is hypothesized that NRC1, required for multiple defense pathways and exerting its activity downstream of EDS1 and upstream of MEK2, is a key regulator of defense.

In chapter 5 different mechanisms generating resistance to pathogens, that operate in plants and in mammals, are discussed. Knowledge of defense pathways and the identification of the genes involved might reveal components required for resistance to many different pathogens.