Authors: Nicholas Rhoades 1,2, Youhuang Xiang 3, Roger W. Innes 3, and Guixia Hao 1
1. USDA, Agricultural Research Service, National Center for Agricultural Utilization Research, Mycotoxin Prevention and Applied Microbiology Research Unit, Peoria, IL
2. Oak Ridge Institute for Science and Education, USDA, Agricultural Research Service, National Center for Agricultural Utilization Research, Mycotoxin Prevention and Applied Microbiology Research Unit, Peoria, IL
3. Department of Biology, Indiana University, Bloomington, IN
Corresponding Author: Guixia Hao, guixia.hao@usda.gov
Presenting Author: Guixia Hao
Abstract
Fusarium head blight (FHB) caused by Fusarium species is a major threat to food safety and security by reducing crop yields and contaminating grains with harmful mycotoxins. RNA interference (RNAi) technology has been widely used to control plant diseases and pests. Two common RNAi application methods are host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS). Each method has its limitations. Therefore, our goal is to develop an alternative RNAi delivery platform using endophytes. Because endophytes colonize their hosts without causing damage, an endophyte-mediated RNAi system is expected to be cost-effective and sustainable. The fungal endophyte Sarocladium zeae has been shown to reduce fungal diseases in corn and wheat. To enhance S. zeae’s ability to reduce FHB and toxin contamination, we investigated whether S. zeae can deliver RNA silencing signals to F. graminearum. As a proof of concept, we expressed a hairpin RNAi construct in S. zeae 34560 that targeted a green fluorescent protein (GFP) gene (hereafter (SzGFPi). We found that SzGFPi strains suppressed GFP expression in a transgenic F. graminearum strain expressing GFP when co-cultured on plates and in liquid. RT-PCR and Northern blot analyses showed that SzGFPi strains produced GFP double stranded RNA (GFP-dsRNA) but did not process them to small interfering RNA (siRNA). Furthermore, we showed that GFP-dsRNA was present in conditioned media in which the Sz34560 GFP-RNAi strains had been grown, indicating that SzGFPi can secrete GFP-dsRNA to reduce GFP expression in F. graminearum. In addition, genome analysis revealed that S. zeae contains some RNA-induced silencing complex (RISC) members, including two Argonaute-like proteins (AGO1 and AGO2), one Dicer-like protein, and at least one RNA-dependent RNA Polymerase (RdRP). Further investigations are underway to determine if the dsRNA delivered by S. zeae can be processed into siRNA in plants and in F. graminearum.
This material is based upon work supported by the U.S. Department of Agriculture. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture.