Location: Cereal Crops Research2011 Annual Report
1a. Objectives (from AD-416)
1) The identification of HST-host gene interactions using purified toxins and wheat mapping populations; 2) Identification of proteinaceous toxin genes using purified toxins in conjunction with mass spec analysis to identify candidate genes for further evaluation; 3) Verification of candidate genes using heterologous expression, transformation, and site directed gene disruption; and 4) Mode of actions studies to identify the molecular and biochemical mechanism whereby the toxin is instrumental in causing disease, including protein-protein interaction studies, inhibitor studies, and protein localization studies.
1b. Approach (from AD-416)
Stagonospora nodorum will be used to produce extracts of the proteinaceous host-selective toxins (HSTs) that we have identified. HSTs will be purified and active fractions will be used to identify protein sequences using mass-spectrometry. Each of the presently characterized toxins has been shown to be a protein that interacts with a corresponding host sensitivity gene in an inverse gene-for-gene manner. Candidate genes will be revealed using the S. nodorum genome sequence. Additionally, bioinformatics will be used to scan the S. nodorum genome to identify candidate genes based on signal sequence domains and predicted protein size in addition to other relevant criteria. We have isolated a nonpathogenic strain of S. nodorum that appears to secrete no toxins. We will use this strain to verify toxin gene candidates. Candidate genes will initially be expressed in this isolate to verify toxin production and pathogenicity changes. Lastly, we will continue to identify new toxins using an international S. nodorum collection. This proposal will enhance economic opportunities for agricultural producers. This will be accomplished by providing valuable information to scientists including breeders for the improvement of wheat as a food crop especially as it relates to providing durable resistance sources to growers.
3. Progress Report
Stagonospora nodorum blotch (SNB) caused by the necrotrophic fungal pathogen S. nodorum is a destructive disease throughout the wheat growing regions of 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 but not on lines lacking Snn1. Avirulent S. nodorum isolates lacking SnTox1 were transformed with the SnTox1 gene, resulting in strains that were virulent on Snn1 wheat lines. Using site directed 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 wheat 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 in order 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 as 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.