Recipient Organization
RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY
3 RUTGERS PLZA
NEW BRUNSWICK,NJ 08901-8559
Performing Department
Plant Biology
Non Technical Summary
This project encompasses two aspects of the Epichloë-grass symbiosis, both of which have potential beneficial impacts on agriculture.Firstly, Endophyte infected grasses generally confer numerous benefits to their host grasses. However, because of the specificity of the interactions, the ability to manipulate the endophyte isolate -host plant combination is limited. Better understanding of the biological basis of the specificity may lead to the ability to produce designer combinations in which Epichloë isolates possessing specific desirable traits can be introduced into plant species that normally do not harbor that Epichloë sp.Next, we are investigating the E. festucae antifungal protein as a likely component of the field level dollar spot resistance observed in strong creeping red fescue. Understanding the basis of dollar spot resistance in endophyte-infected fine fescues may suggest new approaches to dollar spot management in other grass species.Current management practices involve heavy use of expensive fungicides. This project directly addresses the hypothesis that an endophyte antifungal protein may be a component of the dollar spot resistance observed in endophyte-infected red fescues. If so, then it may be possible to utilize this protein in direct applications to protect other grass species, for example creeping bentgrass, from dollar spot disease. This will reduce the use of expensive fungicides, thus reducing turf grass maintenance costs and reducing exposure to toxic chemicals.
Animal Health Component
0%
Research Effort Categories
Basic
60%
Applied
0%
Developmental
40%
Goals / Objectives
One hypothesis of this proposal is that the E. festucae antifungal protein is an effector protein important in the establishment of the symbiotic interaction. We plan to expand on this by examining other small secreted protein genes for possible importance in the symbiosis by systematically knocking out those genes using the CRISPR-cas9 system. The other hypothesis of the proposal is that the E. festucae antifungal protein could be utilized directly as a fungicide to protect plants from dollar spot disease. That knowledge may lead to new approaches for dollar spot management in other grass species.
Project Methods
Systematic knockout of E. festucae candidate effector genesWe will use the CRISPR-cas9 system, which we successfully used for generating the antifungal protein gene knockouts, for the systematic knockout of candidate effector genes. One hundred and forty-one candidate effector protein genes were identified in the E. festucae genome (Hassing et al., 2019). We will prioritize genes for knockout based on differential expression of genes identified in a previous transcriptome study of choke disease in strong creeping red fescue plants infected with E. festucae (Wang et al., 2019). In some symbiotic interactions, during development of the flowering tiller, the normally asymptomatic intercellular fungal mycelium proliferates extensively and eventually forms an external stroma, the fungal fruiting structure that envelops the developing inflorescence. Because the development of the inflorescence is physically constrained by the fungus, this syndrome is referred to as "choke disease". The transcriptome analysis revealed that 19 candidate effector genes were more highly expressed in the asymptomatic inflorescence tissue than in the choke stroma tissue. One of the 19 was the antifungal protein, which we now hypothesize is also an effector protein. We will prioritize for knockouts the candidate effector proteins overexpressed in the asymptomatic inflorescence tissue since in that tissue the fungus is in close contact with the plant tissue, whereas in the choke stroma tissue, most of the fungal tissue is not in contact with the plant. The knockout isolates will then be inoculated into endophyte-free fine fescue plants to assess the impact of the knockout on the symbiosis, as was done with the antifungal protein gene knockouts.Testing if the E. festucae antifungal protein can be used directly to protect plants from diseaseOur hypothesis is that the E. festucae antifungal protein is a factor in the well-documented disease resistance seen in endophyte-infected fine fescues in the field (Clarke et al., 2006). We have demonstrated that the purified protein has activity against the dollar spot fungus in plate assays. The ultimate goal of this research is to determine if the endophyte antifungal protein can protect creeping bentgrass plants from dollar spot disease so that it could be used as an alternative or supplement to synthetic fungicides. To do this requires a substantial amount of purified antifungal protein, which is why we have been focused on testing different expression systems. The Penicillium chrysogenum expression system is our preferred system since it is simpler than the previously used Pichia pastoris system. We have also established greenhouse inoculation protocols for dollar spot. We will test if application of the purified antifungal protein directly to plants can protect them from disease. The purified antifungal protein will be applied to endophyte-free fine fescue and creeping bentgrass plants prior to inoculation, at the same time, and post-inoculation with the dollar spot fungus. If the greenhouse assays are positive we will plan on establishing a field test of the antifungal protein on creeping bentgrass infected with dollar spot. The results of these assays will reveal the potential of the E. festucae antifungal protein as a new means of protecting creeping bentgrass from dollar spot infection. If direct application of the antifungal protein to creeping bentgrass is effective in reducing the disease symptoms of dollar spot it could be developed as an alternative to synthetic fungicides.