Small Cationic Cysteine-Rich Defensin-Derived Antifungal Peptide Controls White Mold in Soybean.

White mold disease caused by a necrotrophic ascomycete pathogen Sclerotinia sclerotiorum results in serious economic losses of soybean yield in the USA. Lack of effective genetic resistance to this disease in soybean germplasm and increasing pathogen resistance to fungicides makes white mold difficult to manage. Small cysteine-rich antifungal peptides with multi-faceted modes of action possess potential for development as sustainable spray-on bio-fungicides. We have previously reported that GMA4CG_V6 peptide, a 17-amino acid variant of the MtDef4 defensin-derived peptide GMA4CG containing the active γ-core motif, exhibits potent antifungal activity against the gray mold fungal pathogen Botrytis cinerea in vitro and in planta. GMA4CG_V6 exhibited antifungal activity against an aggressive field isolate of S. sclerotiorum 555 in vitro with an MIC value of 24 µM. At this concentration, internalization of this peptide into fungal cells occurred prior to discernible membrane permeabilization. GMA4CG_V6 markedly reduced white mold disease symptoms when applied to detached soybean leaves, pods, and stems. Its spray application on soybean plants provided robust control of this disease. GMA4CG_V6 at sub-lethal concentrations reduced sclerotia production. It was also non-phytotoxic to soybean plants. Our results demonstrate that GMA4CG_V6 peptide has potential for development as a bio-fungicide for white mold control in soybean.

Plant defensin MtDef4-derived antifungal peptide with multiple modes of action and potential as a bio-inspired fungicide.

Chemical fungicides have been instrumental in protecting crops from fungal diseases. However, increasing fungal resistance to many of the single-site chemical fungicides calls for the development of new antifungal agents with novel modes of action (MoA). The sequence-divergent cysteine-rich antifungal defensins with multisite MoA are promising starting templates for design of novel peptide-based fungicides. Here, we experimentally tested such a set of 17-amino-acid peptides containing the γ-core motif of the antifungal plant defensin MtDef4. These designed peptides exhibited antifungal properties different from those of MtDef4. Focused analysis of a lead peptide, GMA4CG_V6, showed that it was a random coil in solution with little or no secondary structure elements. Additionally, it exhibited potent cation-tolerant antifungal activity against the plant fungal pathogen Botrytis cinerea, the causal agent of grey mould disease in fruits and vegetables. Its multisite MoA involved localization predominantly to the plasma membrane, permeabilization of the plasma membrane, rapid internalization into the vacuole and cytoplasm, and affinity for the bioactive phosphoinositides phosphatidylinositol 3-phosphate (PI3P), PI4P, and PI5P. The sequence motif RRRW was identified as a major determinant of the antifungal activity of this peptide. While topical spray application of GMA4CG_V6 on Nicotiana benthamiana and tomato plants provided preventive and curative suppression of grey mould disease symptoms, the peptide was not internalized into plant cells. Our findings open the possibility that truncated and modified defensin-derived peptides containing the γ-core sequence could serve as promising candidates for further development of bio-inspired fungicides.

Identification of Indole-3-Acetic Acid-Regulated Genes in Pseudomonas syringae pv. tomato Strain DC3000.

The auxin indole-3-acetic acid (IAA) is a plant hormone that not only regulates plant growth and development but also plays important roles in plant-microbe interactions. We previously reported that IAA alters expression of several virulence-related genes in the plant pathogen Pseudomonas syringae pv. tomato strain DC3000 (PtoDC3000). To learn more about the impact of IAA on regulation of PtoDC3000 gene expression, we performed a global transcriptomic analysis of bacteria grown in culture, in the presence or absence of exogenous IAA. We observed that IAA repressed expression of genes involved in the type III secretion (T3S) system and motility and promoted expression of several known and putative transcriptional regulators. Several of these regulators are orthologs of factors known to regulate stress responses and accordingly expression of several stress response-related genes was also upregulated by IAA. Similar trends in expression for several genes were also observed by quantitative reverse transcription PCR. Using an Arabidopsis thaliana auxin receptor mutant that accumulates elevated auxin, we found that many of the P. syringae genes regulated by IAA in vitro were also regulated by auxin in planta. Collectively the data indicate that IAA modulates many aspects of PtoDC3000 biology, presumably to promote both virulence and survival under stressful conditions, including those encountered in or on plant leaves. IMPORTANCE Indole-3-acetic acid (IAA), a form of the plant hormone auxin, is used by many plant-associated bacteria as a cue to sense the plant environment. Previously, we showed that IAA can promote disease in interactions between the plant pathogen Pseudomonas syringae strain PtoDC000 and one of its hosts, Arabidopsis thaliana. However, the mechanisms by which IAA impacts the biology of PtoDC3000 and promotes disease are not well understood. Here, we demonstrate that IAA is a signal molecule that regulates gene expression in PtoDC3000. The presence of exogenous IAA affects expression of over 700 genes in the bacteria, including genes involved in type III secretion and genes involved in stress response. This work offers insight into the roles of auxin-promoting pathogenesis.

Gene expression and evidence of coregulation of the production of some metabolites of chilli pepper inoculated with Pectobacterium carotovorum ssp. carotovorum.

Chilli pepper (Capsicum annuum L.) is susceptible to Pectobacterium carotovorum subsp. carotovorum (Pcc), the causal agent of soft rot disease in crops. Understanding the molecular principles of systemic acquired resistance, which is poorly understood in chilli pepper, represents an important step towards understanding inducible defence responses and can assist in designing appropriate intervention strategies for crop disease management. Accordingly, we investigated (via real-time PCR and metabolomics profiling) the molecular response of chilli pepper to Pcc by characterisation of the crucial metabolic regulators involved in the establishment of defence response. We profiled 13 key inducible defence response genes, which included MYB transcriptor factor, ethylene response element-binding protein, suppressor of the G2 allele of Skp1, cytochrome P450, small Sar1 (GTPase), hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase, pathogenesis-related protein 1a, endo-1,3-ß-glucanase, chitinase, proteinase inhibitor, defensin, coiled-coil-nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR) resistance and phenylalanine ammonia lyase. In addition, we determined metabolomic shifts induced by Pcc in pepper. The PCR results revealed a significant induction of the selected plant defence-related genes in response to Pcc inoculation; the metabolomic profiling showed that of 99 primary metabolites profiled the quantities of acetylcarnitine, adenosine, adenosine 3',5' cyclic monophosphate, guanosine 3',5' cyclic monophosphate and inosine decreased in pepper leaves inoculated with Pcc.