Location: Cereal Crops Research2011 Annual Report
1a. Objectives (from AD-416)
Objective 1. Evaluate the importance of HST-host gene interactions identified using culture filtrates and purified toxins in conjunction with wheat mapping populations including the development of differential wheat lines for each toxin. Objective 2. Identify candidate HSTs by mass spec analysis of purified active protein preparations and by using a bioinformatics gene selection approach. Objective 3. Verify candidate genes using heterologous expression, transformation, and site directed gene disruption. Objective 4. Characterize the function and mode of action of SnTox3 to identify the molecular and biochemical mechanism whereby the toxin is effective in inducing disease.
1b. Approach (from AD-416)
We have established that the Stagonospora nodorum-wheat interaction consists of pathogen produced effector proteins i.e. host selective toxins (HSTs) that interact with dominant host gene products to induce disease. This interaction is similar to a classical gene-for-gene interaction except that host recognition of the effector proteins leads to susceptibility rather than resistance and therefore acts in an inverse gene-for-gene manner. We have published or submitted for publication five of these HST – host gene interactions. Additionally, we have accumulated evidence for four more interactions. Each of these nine interactions involves a single proteinaceous HST that interacts directly or indirectly with a dominant host gene product leading to disease. In this proposal we would like to 1) characterize the newly identified interactions, 2) clone the associated HST encoding genes from the pathogen for further analysis of individual interactions, and 3) do functional analysis and evaluate the mode of action of SnTox3, a recently cloned HST involved in the SnTox3-Snn3 interaction.
3. Progress Report
Stagonospora nodorum blotch (SNB) caused by the necrotrophic fungal pathogen S. nodorum is a destructive disease of wheat throughout the world. We have shown that S. nodorum produces a suite of necrotrophic effectors that each interact with specific host sensitivity gene products to induce disease on wheat. SnTox1 was recently cloned and in the previous year we have characterized the interaction of SnTox1 with its corresponding sensitivity gene Snn1. Validation of the gene was accomplished by heterologous expression in Pichia pastoris, transformation and expression in an avirulent isolate of S. nodorum, and site directed gene disruption in virulent isolates carrying a functional SnTox1 gene. Culture filtrates of P. pastoris expressing SnTox1 induced necrosis only on lines harboring the Snn1 gene Avirulent S. nodorum isolates lacking SnTox1 were transformed with the SnTox1 gene, resulting in strains that were virulent on Snn1 wheat lines. Using gene disruption, SnTox1 was eliminated in virulent isolates and the level of disease was significantly reduced on lines harboring Snn1. However, disease levels were not changed on lines lacking the Snn1 gene. Characterization of the SnTox1-Snn1 interaction showed that the development of necrosis due to the SnTox1-Snn1 interaction is dependent on light and that light is necessary for disease development involving the SnTox1-Snn1 interaction. Although the SnTox1-Snn1 interaction results in susceptibility, several hallmarks of a host resistance response during this interaction are present. These include an oxidative burst identified by an increased accumulation of hydrogen peroxide, up regulation of pathogenesis related (PR) gene expression in the host, and DNA laddering, a typical apoptotic response in both plant and animal resistance responses. Additionally, initiation of candidate gene expression from a bioinformatically assembled list of candidate effector genes has revealed a necrotrophic effector responsible for a disease QTL in wheat associated with wheat chromosome 3A. This QTL was reported in a previous publication from our lab, but until now it was not known that an effector sensitivity gene was responsible for this QTL. Additional prioritized candidates are being evaluated to identify the cause of many of the disease QTLs in segregating wheat populations. The work on the S. nodorum-wheat interaction has already led to a better understanding of how this and other necrotrophic pathogens are causing disease. This research has also provided developers and breeders with critical knowledge needed for the development of more resistant varieties. One of the main outcomes of this research is the knowledge that necrotrophic pathogens may be using aspects of the host resistance mechanism to induce disease shown by the hallmarks of a classical resistance response during susceptibility. The fact that this necrotrophic system involves active (genetically dominant) susceptibility genes rather than active resistance is critical knowledge for developing increased disease resistant germplasm. ADODR monitoring included personal visitation, email correspondence, and telephone discussions.