Polygalacturonase-inhibiting proteins (PGIPs) are plant defence molecules inhibiting the activity of fungal endo-polygalacturonases (endo-PGs). We found that soybean and bean PGIPs inhibited the endo-PG activity produced by the isolate FC-10 of Fusarium moniliforme but not the enzyme activity produced by the isolate PD of F. moniliforme. The bean PGIP proved to be ineffective against all the PG isoforms produced by the PD isolate. Deduced amino acid sequence comparison between PGs from PD, FC-10 and 62264 isolates identified the structural regions of the enzyme possibly related to its resistance to PGIP inhibition. These include one region at the N-terminal portion of the enzyme and a few single amino acid substitutions along the entire sequence, two of which surrounding the active site.

Grapevine is an economically important crop, and the recent completion of its genome makes it possible to study the function of specific genes through reverse genetics. However, the analysis of gene function by RNA interference (RNAi) in grapevine is difficult, because the generation of stable transgenic plants has low efficiency and is time consuming. Recently, transient expression of genes in grapevine leaves has been obtained by Agrobacterium tumefaciens infiltration (agroinfiltration). We therefore tested the possibility to silence grapevine genes by agroinfiltration of RNAi constructs. A construct to express a double strand RNA (dsRNA) corresponding to the defense-related gene VvPGIP1, encoding a polygalacturonase-inhibiting protein (PGIP), was obtained and transiently expressed by agroinfiltration in leaves of grapevine plants grown in vitro. Expression of VvPGIP1 and accumulation of PGIP activity were strongly induced by infiltration with control bacteria, but not with bacteria carrying the dsRNA construct, indicating that the gene was efficiently silenced. In contrast, expression of another defense-related gene, VST1, encoding a stilbene synthase, was unaffected by the dsRNA construct. We have therefore demonstrated the possibility of transient down-regulation of grapevine genes by agroinfiltration of constructs for the expression of dsRNA. This system can be employed to evaluate the effectiveness of constructs that can be subsequently used to generate stable RNAi transgenic plants.

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Fusarium graminearum is a toxigenic fungal pathogen that causes Fusarium head blight (FHB) and crown rot on cereal crops worldwide. This fungus also causes damping-off and crown and root rots at the early stage of crop development in soybean cultivated in North and South America. Several F. graminearum genes were investigated for their contribution to FHB in cereals but no inherent study is reported for the dicotyledonous soybean host. In this study we determined the disease severity on soybean seedlings of five single gene disrupted mutants of F. graminearum, previously characterized in wheat spike infection. Three of these mutants are impaired on a specific function as the production of deoxynivalenol (DON, Δtri5), lipase (ΔFgl1), and xylanase (Δxyl03624), while the remaining two are MAP kinase mutants (ΔFgOS-2, Δgpmk1), which are altered in signaling pathways. The mutants that were reduced in virulence (Δtri5, ΔFgl1, and ΔFgOS-2) or are avirulent (Δgpmk1) on wheat were correspondently less virulent or avirulent in soybean seedlings, as shown by the extension of lesions and seedling lengths. The Δxyl03624 mutant was as virulent as the wild type mirroring the behavior observed in wheat. However, a different ranking of symptom severity occurred in the two hosts: the ΔFgOS-2 mutant, that infects wheat spikelets similarly to Δtri5 and ΔFgl1 mutants, provided much reduced symptoms in soybean. Differently from the other mutants, we observed that the ΔFgOS-2 mutant was several fold more sensitive to the glyceollin phytoalexin suggesting that its reduced virulence may be due to its hypersensitivity to this phytoalexin. In conclusion, lipase and DON seem important for full disease symptom development in soybean seedlings, OS-2 and Gpmk1 MAP kinases are essential for virulence, and OS-2 is involved in conferring resistance to the soybean phytoalexin.

Endo-polygalacturonases (PGs) and xylanases have been shown to play an important role during pathogenesis of some fungal pathogens of dicot plants, while their role in monocot pathogens is less defined. Pg1 and xyr1 genes of the wheat pathogen Fusarium graminearum encode the main PG and the major regulator of xylanase production, respectively. Single- and double-disrupted mutants for these genes were obtained to assess their contribution to fungal infection. Compared with wild-type strain, the ∆pg mutant showed a nearly abolished PG activity, slight reduced virulence on soybean seedlings, but no significant difference in disease symptoms on wheat spikes; the ∆xyr mutant was strongly reduced in xylanase activity and moderately reduced in cellulase activity but was as virulent as wild type on both soybean and wheat plants. Consequently, the ΔpgΔxyr double mutant was impaired in xylanase, PG, and cellulase activities but, differently from single mutants, was significantly reduced in virulence on both plants. These findings demonstrate that the concurrent presence of PG, xylanase, and cellulase activities is necessary for full virulence. The observation that the uronides released from wheat cell wall after a F. graminearum PG treatment were largely increased by the fungal xylanases suggests that these enzymes act synergistically in deconstructing the plant cell wall.