Performing Department
Plant and Microbial Biology
Non Technical Summary
The powdery disease is a growing threat to tomato production worldwide, leading to losses in yield and fruit quality, as well as diversion of grower capital and labor to fight this disease. We will use newly available genome sequence information from the tomato powdery mildew fungal pathogen to identify sources of powdery mildew disease resistance that can be deployed by breeders. One of the weapons pathogens use to disarm plant defenses is a group of diverse effector proteins. The powdery mildews appear to use between 100 and 600 of such effectors to manipulate their plant hosts to the benefit of the pathogen. We will screen for tomato powdery mildew effectors that modify plant defenses in rapid high through-put assays. Those effectors with defense-modifying activity will be used as bait to identify the plant defenses that are the effectors' targets. We expect to identify classic resistance proteins as well as novel forms of resistance. These resistances will be introduced into susceptible tomato varieties that will be grown under disease conditions to verify the efficacy of the resistances identified in our experiments. This work will augment traditional breeding, where bottlenecks in identifying new sources powdery mildew resistance often occur. Additionally, the powdery mildew resistant lines generated in the final stage of this project will serve as proof-of-concept for our strategy to rapidly identify and deploy disease resistance genes and will serve as a model for disease resistance genes discovery in other crop species for other diseases. ?
Animal Health Component
0%
Research Effort Categories
Basic
50%
Applied
20%
Developmental
30%
Goals / Objectives
Powdery mildew disease, caused by the fungal pathogen Pseudoidium neolycopersici (Pnl) represents a growing threat to tomato production both in greenhouse and in field grown varieties. Disease losses incur additional costs beyond lost product for growers, including labor, water, fertilizer and energy costs of raising the diseased tomato plants. Fungicide treatments are the principle means of powdery mildew control. As mentioned above, limited natural sources of resistance to tomato powdery mildew have been identified. This paucity of resources for plant breeders to draw upon in developing powdery mildew resistant cultivars motivates our work. Our proposed work is expected to lead to the identification of a substantial increase in genes conferring resistance to powdery mildew and thereby expand options for stacking resistance to create stable, robust and broad-spectrum resistance. These will serve as a panel of options for plant breeders and genetic engineers looking to incorporate powdery mildew resistance traits into new cultivars to draw upon. Our work should also lead to reductions in the need for fungicide applications to control powdery mildew. Furthermore, the methods we develop can be utilized in the future studies to identify new powdery mildew disease resistance genes, without relying on genetic crosses, as such crosses can be difficult or in some cases impossible to perform. Finally, the strategy we have outlined can be adapted for use with other pathogens and other crop species to accelerate the identification and deployment of new resistance genes.Our working hypothesis is that the tomato proteins, which are powdery mildew effector targets, represent potential new sources of disease resistance. Since the first identification of pathogen effectors >20 years ago, the research community that many effector proteins are delivered into host plant cells, where they modify host functions to the benefit of the pathogen. Some effectors act modify host defenses (e.g., suppress reactive oxygen species production) and some act to promote plant functions that support pathogen growth (e.g., enhance the expression of plasma membrane localized sugar exporters). In this project, we will identify those powdery mildew effectors that modify tomato defenses and the tomato defense-related proteins that the effectors target. Estimates of the number of effectors in powdery mildews range from ~100 in Golovinomyces orontii (Arabidopsis powdery mildew) to ~600 in Blumeria graminis f. sp. tritici (wheat powdery mildew). These large numbers suggests that we need to apply high through-put methods to identify Pnl effector proteins that manipulate tomato defenses. We will take two approaches to screen for effectors that modify tomato defenses. In one, we will screen for powdery mildew effectors that trigger a classic hypersensitive resistance response when introduced into a resistant tomato accession (i.e., effectors that trigger ETI, Effector Triggered Immunity). In the second approach, we will screen for powdery mildew effectors that suppress PTI, Pattern Triggered (or Basal) Immunity. The host proteins involved in either ETI or PTI and that are targeted by powdery mildew effectors for modification will used in the latter stages of the project to generate powdery mildew-resistant tomato lines.Our aims to address this hypothesis include:1. Identify candidate Pnl effector genes and candidate tomato reporter genes.2. Identify Pnl effectors that manipulate plant defenses (i.e., PTI or ETI).3. Identify tomato target proteins for these Pnl effectors4. Validate tomato target proteins in powdery mildew disease assays in stably transformed tomato plants grown in typical greenhouse conditions.
Project Methods
Aim 1. Identify candidate Pnl effector genes and candidate tomato reporter genes.The methods used to achieve this aim will include bioinformatics analyses of the tomato powdery mildew genome sequence to identify genes encoding candidate effector proteins. This approach will be coupled with transcriptome profiling of powdery mildew infected tomato leaves to determine which effector genes are expressed while the powdery mildew pathogen is successfully infecting tomato leaves. The transcriptome data will also be mined for tomato genes that can be used as reporters of the activation of basal plant defenses that occurs during infections. Specifically, tomato genes that are highly expressed when basal plant defenses are elicited by chitin (a component of powdery mildew cell walls) but not in powdery mildew-infected tomato (i.e., due to the suppression of basal defenses by powdery mildew effector proteins) will be developed into reporters (i.e., promoter:LUC clones). These reporters will be used to screen the repertoire of powdery mildew effectors for those that suppress basal defenses and reporter gene expression in tomato.Aim 2. Identify Pnl effectors that manipulate plant defenses (i.e., PTI or ETI).Screen for effectors eliciting ETI: The candidate effector genes from Aim1 will cloned into a transient expression system and each effector will be individually expressed in the leaflets of the wild powdery mildew resistant accession Solanum peruvianum LA2172. At daily intervals the treated leaflets will be assessed for the presence of a hypersensitive resistance response indicative of the interaction of the effector with a specific powdery mildew resistance protein. Up to 5 of these effectors will be advanced to Aim 3.Screen for effectors suppressing PTI: The candidate effector genes will cloned into a transient expression vector as above. These vectors will be introduced into tomato leaflets expressing one of five tomato reporter genes developed in Aim 1. The treated leaflets will be photographed to detect the level of LUC expression for each of the reporter genes. Up to 5 effectors that suppress the expression of the reporter genes will be advanced to Aim 3.Aim 3. Identify tomato target proteins for these Pnl effectorsThe yeast-two hybrid method will be the primary method used to identify the specific tomato proteins that are the target of the 10 powdery mildew effectors selected in Aim 2. Based on prior literature, about 20 to 40 host proteins will be identified this this method using the 10 effector proteins from Aim 2. Genes encoding the 10 host proteins that show the strongest level of protection against powdery mildew in transient expression assays will be advanced to Aim 4.Aim 4. Validate tomato target proteins in powdery mildew disease assays in stably transformed tomato plants grown in typical greenhouse conditions.The stably transformed tomato lines will also be assessed for their growth habit and fruit characteristics to ensure the introduced genes do not have undesired pleiotropic effects with the guidance of plant breeder, Dr. F. Hempel. The stably transformed lines will be produced in Dr. Li's laboratory (Chinese Academy of Sciences, Beijing, China) using methods that are well developed in his laboratory. We will travel to his lab to perform the powdery mildew disease assays and assessments of the transformants. The stably transformed tomato lines will be returned to our laboratory in Berkeley, CA and assessed for powdery mildew resistance against California strains of the tomato powdery mildew and performance characteristics as well.