Validamycin A Enhances the Interaction Between Neutral Trehalase and 14-3-3 Protein Bmh1 in Fusarium graminearum.

In agriculture, Trehalase is considered the main target of the biological fungicide validamycin A, and the toxicology mechanism of validamycin A is unknown. 14-3-3 proteins, highly conserved proteins, participate in diverse cellular processes, including enzyme activation, protein localization, and acting as a molecular chaperone. In Saccharomyces cerevisiae, the 14-3-3 protein Bmh1could interact with Nth1 to respond to specific external stimuli. Here, we characterized FgNth, FgBmh1, and FgBmh2 in Fusarium graminearum. ΔFgNth, ΔFgBmh1, and ΔFgBmh2 displayed great growth defects and their peripheral tips hyphae generated more branches when compared with wild-type (WT) PH-1. When exposed to validamycin A as well as high osmotic and high temperature stresses, ΔFgNth, ΔFgBmh1, and ΔFgBmh2 showed more tolerance than WT. Both ΔFgNth and ΔFgBmh1 displayed reduced deoxynivalenol production but opposite for ΔFgBmh2, and all three deletion mutants showed reduced virulence on wheat coleoptiles. In addition, coimmunoprecipitation (Co-IP) experiments suggested that FgBmh1 and FgBmh2 both interact with FgNth, but no interaction was detected between FgBmh1 and FgBmh2 in our experiments. Further, validamycin A enhances the interaction between FgBmh1 and FgNth in a positive correlation under concentrations of 1 to 100 µg/ml. In addition, both high osmotic and high temperature stresses promote the interaction between FgBmh1 and FgNth. Co-IP assay also showed that neither FgBmh1 nor FgBmh2 could interact with FgPbs2, a MAPKK kinase in the high-osmolarity glycerol pathway. However, FgBmh2 but not FgBmh1 binds to the heat shock protein FgHsp70 in F. graminearum. Taken together, our results demonstrate that FgNth and FgBmh proteins are involved in growth and responses to external stresses and virulence; and validamycin enhanced the interaction between FgNth and FgBmh1in F. graminearum.

A ß2-tubulin dsRNA derived from Fusarium asiaticum confers plant resistance to multiple phytopathogens and reduces fungicide resistance.

Crops are attacked by a large number of pathogens which are responsible for an approximately 30% loss in global crop production at pre- and post-harvest levels. In light of the continuing emergence of fungicide resistance, the needs for new agricultural drugs turn out to be much more critical. Here we demonstrated a Faß2Tub-3 dsRNA derived from Fusarium asiaticum had broad-spectrum antifungal activity against Fusarium spp., Botrytis cinerea, Magnaporthe oryzae and Colletotrichum truncatum, with an additional function of reducing the dosage of carbendazim (MBC) fungicide. RNAi molecules derived from different regions of ß2-tubulin gene had different effects on mycelial growth, asexual reproduction and virulence. Faß2Tub-3 (one of ß2-tubulin segments) exhibited a strong silencing efficacy both on ß1-tubulin and ß2-tubulin genes in F. asiaticum. Faß2Tub-3 sequence was found to be highly conserved among Fusarium spp., Botrytis cinerea, Magnaporthe oryzae and Colletotrichum truncatum. The Faß2Tub-3 dsRNA demonstrated a broad-spectrum antifungal activity against these fungi in vitro and on living plant. More importantly, Faß2Tub-3 dsRNA increased the fungal sensitivity to MBC, while MBC increased the duration of Faß2Tub-3 dsRNA. Our findings suggest a new anti-fungal agent (Faß2Tub-3 dsRNA) for plant protection against diverse pathogens and for fungicide reduction.

A myosin5 dsRNA that reduces the fungicide resistance and pathogenicity of Fusarium asiaticum.

Fungal resistance to fungicides is a serious challenge in crop protection. Although strategies have been found to prevent the development of fungicide resistance, rare strategy has been found to quickly reduce such resistance once it has occurred. We demonstrate that the application of dsRNAs, which inhibit the expression of the phenamacril (fungicide JS399-19) target gene-Myosin 5 (Myo5) in Fusarium, decreased F. asiaticum resistance to phenamacril and infection. RNAi molecules derived from different regions of Myo5 gene had different effects on phenamacril-resistance. Myo5-8 (one of Myo5 segments) exhibited great and stable effect on phenamacril-resistant reduction both in vivo and in vitro. Myo5 mRNA and protein were both reduced when mycelium was treated with Myo5-8 dsRNA. After a mixture of Myo5-8 dsRNA and phenamacril treatment, plants can highly control the infection of phenamacril-resistant strain. The antifungal activity of Myo5-8 dsRNA plus phenamacril effected longer than a single Myo5-8 dsRNA. In addition, no off-target sequences were found in wheat and/or other plant and animal species for Myo5-8 dsRNA sequence. Our findings suggest a new strategy for fungicide resistant reduction and for designing new fungicides to control pathogens which easily develop fungicide resistance.

Secondary amplification of siRNA machinery limits the application of spray-induced gene silencing.

Spray-induced gene silencing (SIGS) is an innovative strategy for crop protection. However, the mechanism of SIGS is not known. Here, we first demonstrate that secondary small interfering RNA (siRNA) amplification limits the application of SIGS. A myosin5 gene (Myo5) was chosen as the target of SIGS in an agronomically important pathogen-Fusarium asiaticum. Five segments corresponding to the different regions of the Myo5 gene were found to efficiently silence Myo5, resulting in cell wall defects, life cycle disruption and virulence reduction. Myo5-8 (one of the Myo5 segments) induced sequence-specific RNA interference (RNAi) activity in F. asiaticum, F. graminearum, F. tricinctum and F. oxysporum, but not in other fungi, in vitro. Remarkably, the silencing of Myo5 lasted for only 9 h unless the double-stranded RNA (dsRNA) was continuously supplied, because F. asiaticum is unable to maintain siRNA amplification. After spraying on plants, dsRNAs were more efficiently taken up via the wounded surface. The antifungal activity of dsRNAs taken up by plant cells was higher and longer lasting than that dried onto the plant surface. In contrast with dsRNAs in fungi, dsRNAs in plant cells could efficiently turn into substantial siRNAs via secondary amplification machinery. Our findings provide new implications to develop SIGS as a mainstream disease control strategy against Fusarium and other fungi.