Ambika Pokhrel

Frogeye Leaf Spot disease, caused by a fungus Cercospora sojina, is a serious disease affecting soybean production. Its control is challenged by resistance to Quinone outside Inhibitor (QoI) fungicides, but plant antifungal peptides with multi-faceted modes of action (MoA) offer a promising alternative. Here, we showed that chickpea nodule-specific cysteine-rich peptide, NCR13_Peptide Folding Variant1 (NCR13_PFV1), exhibited antifungal activity against QoI-sensitive and -resistant field isolates of C. sojina at nanomolar concentrations representing the first antifungal NCR peptide reported effective against C. sojina. Spray-application of this peptide showed no phytotoxicity and effectively protected soybean against FLS. When combined with the QoI fungicide azoxystrobin, NCR13_PFV1 provided additive control of FLS. NCR13_PFV1 disrupted plasma membrane, induced reactive oxygen species (ROS) production, and was rapidly internalized, localizing in the nucleus. It bound to fungal ribosomal RNA and inhibited protein translation. RNA-seq studies revealed the upregulation of several genes encoding heme binding proteins in peptide-challenged C. sojina. Notably, iron supplementation in the growth medium reduced the peptide-induced ROS and antifungal activity, revealing the importance of iron homeostasis in protection or recovery of C. sojina from oxidative stress. Overall, NCR13_PFV1 with its multi-faceted MoA holds potential as a bio-fungicide for FLS control, complementing QoI fungicides and overcoming resistance in C. sojina.

Frogeye Leaf Spot (FLS) disease, caused by a fungal pathogen Cercospora sojina , is a serious threat to soybean production globally. The control of FLS is facing a major challenge due to the rapid emergence of pathogen resistance to Quinone outside Inhibitor (QoI) fungicides. Effective long-term management of FLS in soybean calls for the discovery of antifungal compounds with new modes of action (MoA), durability and safety. Here, we showed that chickpea nodule-specific cysteine-rich peptide, NCR13_Peptide Folding Variant1 (NCR13_PFV1), exhibited antifungal activity against QoI-sensitive and -resistant field isolates of C. sojina at nanomolar concentrations representing the first antifungal NCR peptide reported effective against C. sojina . Spray-application of this peptide showed no phytotoxicity and effectively protected soybean against FLS. When combined with the QoI fungicide azoxystrobin, NCR13_PFV1 provided additive control of FLS. NCR13_PFV1 induced plasma membrane disruption and production of reactive oxygen species (ROS) in C. sojina . NCR13_PFV1 was rapidly internalized into fungal cells where it accumulated in the cytoplasm, localized inside nucleus, bound to fungal ribosomal RNA and inhibited protein translation in vivo . RNA-seq studies revealed the upregulation of several genes encoding heme binding proteins in peptide-challenged C. sojina . Notably, iron supplementation in the growth medium reduced the peptide-induced ROS and antifungal activity, revealing the importance of iron homeostasis in protection or recovery of C. sojina from oxidative stress. Overall, NCR13_PFV1 with multiple MoA holds potential as a bio-fungicide for FLS control complementing conventional QoI fungicides and overcoming fungicide resistance in C. sojina .

The Medicago truncatula bi-domain defensin MtDef5 exhibits activity against some fungal pathogens at sub-micromolar concentrations. It is comprised of two single-domain defensins, MtDef5A and MtDef5B, connected with the linker peptide APKKVEP. MtDef5B exhibits greater anti-fungal potency compared with MtDef5A. Here, we identified amino acid residues important for anti-fungal activity of MtDef5B, and determined its modes of action. MtDef5B inhibited spore germination of Botrytis cinerea (Bc) at low micromolar concentrations, but not that of Colletotrichum gloeosporioides (Cg). MtDef5B permeabilized the plasma membrane, induced the production of reactive oxygen species, and localized to the nucleoli in Bc germlings. We selected the carboxy-terminal MtDef5A-derived GMA5AC peptide for mutagenesis because of its lower cationic charge than the corresponding MtDef5B-derived peptide, and found that it inhibited spore germination of Bc, but not of Cg. However, a variant, GMA5AC_V2, did inhibit spore germination of Cg and exhibited a multi-faceted mode of action. Spraying GMA5AC_V2 on the leaves of pepper plants resulted in pre- and post-inoculation control of the gray mold disease caused by Bc. Furthermore, when applied on tomato fruits pre-inoculated with Cg, this peptide reduced the anthracnose disease symptoms. These results highlight the potential of short-chain defensin-derived peptides for management of fungal diseases.

Medicago truncatula bi-domain defensin MtDef5 exhibits antifungal activity at sub-micromolar concentrations against some fungal pathogens. It comprises two single-domain defensins, MtDef5A and MtDef5B, connected with a linker APKKVEP. MtDef5B is a more potent antifungal defensin than MtDef5A. We identified amino acid residues important for antifungal activity of MtDef5B and elucidated its modes of action (MoA). MtDef5B inhibited spore germination of Botrytis cinerea (Bc) at low micromolar concentrations. However, it did not inhibit spore germination of Colletotrichum gloeosporioides (Cg) . MtDef5B permeabilized the plasma membrane, induced reactive oxygen species and traveled to the nucleoli in germlings of Bc . Furthermore, a carboxy-terminal MtDef5A-derived GMA5AC peptide was selected for mutagenesis because of its lower cationicity than the corresponding MtDef5B-derived peptide. GMA5AC inhibited spore germination of Bc , but not of Cg . However, GMA5AC_V2, a variant of GMA5AC, inhibited spore germination of Cg and exhibited multi-faceted MoA. Spray-application of GMA5AC_V2 on the leaves of pepper plants demonstrated preventive and curative control of the gray mold disease. Furthermore, when applied topically on tomato fruits pre-inoculated with the pathogen Cg , this peptide reduced anthracnose disease symptoms. This study highlights the potential of short chain defensin-derived peptides for management of fungal diseases. Highlights: The bi-domain MtDef5-derived antifungal peptides exhibit multiple modes of action and confer resistance against the gray mold and anthracnose diseases in pepper plants and tomato fruits, respectively.

Small cysteine-rich antifungal peptides with multi-site modes of action (MoA) have potential for development as biofungicides. In particular, legumes of the inverted repeat-lacking clade express a large family of nodule-specific cysteine-rich (NCR) peptides that orchestrate differentiation of nitrogen-fixing bacteria into bacteroids. These NCRs can form two or three intramolecular disulfide bonds and a subset of these peptides with high cationicity exhibits antifungal activity. However, the importance of intramolecular disulfide pairing and MoA against fungal pathogens for most of these plant peptides remains to be elucidated. Our study focused on a highly cationic chickpea NCR13, which has a net charge of +8 and contains six cysteines capable of forming three disulfide bonds. NCR13 expression in Pichia pastoris resulted in formation of two peptide folding variants, NCR13_PFV1 and NCR13_PFV2, that differed in the pairing of two out of three disulfide bonds despite having an identical amino acid sequence. The NMR structure of each PFV revealed a unique three-dimensional fold with the PFV1 structure being more compact but less dynamic. Surprisingly, PFV1 and PFV2 differed profoundly in the potency of antifungal activity against several fungal plant pathogens and their multi-faceted MoA. PFV1 showed significantly faster fungal cell-permeabilizing and cell entry capabilities as well as greater stability once inside the fungal cells. Additionally, PFV1 was more effective in binding fungal ribosomal RNA and inhibiting protein translation in vitro. Furthermore, when sprayed on pepper and tomato plants, PFV1 was more effective in reducing disease symptoms caused by Botrytis cinerea, causal agent of gray mold disease in fruits, vegetables, and flowers. In conclusion, our work highlights the significant impact of disulfide pairing on the antifungal activity and MoA of NCR13 and provides a structural framework for design of novel, potent antifungal peptides for agricultural use.

Small cysteine-rich antifungal peptides with multi-site modes of action (MoA) have potential for development as biofungicides. In particular, legumes of the inverted-repeat lacking clade express a large family of nodule-specific cysteine-rich (NCR) peptides that orchestrate differentiation of nitrogen-fixing bacteria into bacteroids. These NCRs can form 2 or 3 intramolecular disulfide bonds and a subset of these peptides with high cationicity exhibits antifungal activity. However, the importance of intramolecular disulfide pairing and MoA against fungal pathogens for most of these plant peptides remains to be elucidated. Our study focused on a highly cationic chickpea NCR13, which has a net charge of +9 and contains six cysteines capable of forming three disulfide bonds. NCR13 expression in Pichia pastoris resulted in formation of two peptide folding variants, NCR13_PFV1 and NCR13_PFV2, that differed in the pairing of two out of three disulfide bonds despite having an identical amino acid sequence. The NMR structure of each PFV revealed a unique three-dimensional fold with the PFV1 structure being more compact but less dynamic. Surprisingly, PFV1 and PFV2 differed profoundly in the potency of antifungal activity against several fungal plant pathogens and their multi-faceted MoA. PFV1 showed significantly faster fungal cell-permeabilizing and cell entry capabilities as well as greater stability once inside the fungal cells. Additionally, PFV1 was more effective in binding fungal ribosomal RNA and inhibiting protein translation in vitro . Furthermore, when sprayed on pepper and tomato plants, PFV1 was more effective in reducing disease symptoms caused by Botrytis cinerea , causal agent of gray mold disease in fruits, vegetables and flowers. In conclusion, our work highlights the significant impact of disulfide pairing on the antifungal activity and MoA of NCR13 and provides structural framework for design of novel, potent antifungal peptides for agricultural use. Author Summary: Fungal pathogens cause significant pre-harvest and post-harvest losses of crop yield, making them a serious biological threat to global food security. Chemical fungicides are effective in controlling fungal diseases across various crops. However, rapid evolution of fungal pathogen resistance to single-site chemical fungicides in agriculture has created an urgent need for development of safe, sustainable, and cost-effective multi-site fungicides. Nodule-specific cysteine-rich (NCR) peptides expressed in the inverted-repeat lacking clade legume plants exhibit potent antifungal activity; however, their modes of action (MoA) are poorly understood. Particularly, the specific contribution of disulfide pairing to the potency and spectrum of antifungal activity against fungal plant pathogens and MoA of these peptides remains to be identified. Chickpea NCR13 expressed in P. pastoris generates two peptide variants that differ in their disulfide cross-linking pattern. These variants exhibit striking differences in their three-dimensional structures and potency of antifungal activity against multiple fungal pathogens and MoA. They also differ in their ability to confer resistance to gray mold in pepper and tomato plants. Our study highlights the major impact a specific pattern of disulfide pairing can have on the in vitro and in planta antifungal activity of an NCR peptide.

The United States is the third largest producer of cotton and the largest exporter of cotton globally. Fusarium wilt, caused by the soilborne fungal pathogen Fusarium oxysporum f. sp. vasinfectum ( Fov), was estimated to cause a $21 million cotton yield loss in 2022. Historically, Alabama was an important producer of cotton in the Southeastern United States and was the first state in which Fusarium wilt on cotton was described. To assess the genetic diversity of Fov field isolates in Alabama, 118 field isolates were collected from six counties across the state from 2014 to 2016. Phylogenetic analysis using TEF1 and RPB2 placed the Fov field isolates into 18 haplotypes. Upon profiling the Tfo1 transposon insertion in the NAT gene, it was determined that no race 4 isolates were recovered in Alabama. Representatives of all field isolate haplotypes caused disease on Upland cotton variety Rowden in a hydroponic test tube assay. Two haplotype A isolates were the most aggressive isolates recovered, and haplotype A isolate TF1 was more aggressive than the race 4 isolate 89-1A on Upland cotton and had similar symptom severity on Pima cotton. Karyotype profiling indicted an abundance of small chromosomes characteristic of karyotypes that include accessory chromosomes, with considerable variability between isolates. Collectively, our study indicates that Fov isolates from Alabama are genetically diverse, which may have been promoted by its persistence in cotton fields.

The Fusarium solani species complex (FSSC) constitutes at least 77 phylogenetically distinct species including several agriculturally important and clinically relevant opportunistic pathogens. As with other Fusaria, they have been well documented to produce many secondary metabolites—compounds that are not required for the fungus to grow or develop but may be beneficial to the organism. An analysis of ten genomes from fungi within the terminal clade (clade 3) of the FSSC revealed each genome encoded 35 (F. cucurbitcola) to 48 (F. tenucristatum) secondary metabolite biosynthetic gene clusters (BGCs). A total of seventy-four different BGCs were identified from the ten FSSC genomes including seven polyketide synthases (PKS), thirteen nonribosomal peptide synthetases (NRPS), two terpene synthase BGCs, and a single dimethylallytryptophan synthase (DMATS) BGC conserved in all the genomes. Some of the clusters that were shared included those responsible for producing naphthoquinones such as fusarubins, a red pigmented compound, squalestatin, and the siderophores malonichrome, ferricrocin, and triacetylfusarinine. Eight novel NRPS and five novel PKS BGCs were identified, while BGCs predicted to produce radicicol, gibberellin, and fusaoctaxin were identified, which have not previously described in members of the FSSC. The diversity of the secondary metabolite repertoire of the FSSC may contribute to the expansive host range of these fungi and their ability to colonize broad habitats.

Fusarium oxysporum f. sp. vasinfectum is an important plant pathogen responsible for vascular wilt disease on cotton. Members of this group are known to carry supernumerary chromosomes that encode virulence factors. We sequenced the genomes of five F. oxysporum f. sp. vasinfectum isolates, including the genome of a representative of the highly virulent genotype race 4, at a high coverage to assemble reference-quality genomes. These genomes provide a necessary resource for comparative genomic analyses to identify genes or genome features that are involved in pathogenicity on cotton and may ultimately be used to identify improved management strategies.