Antimicrobial peptides (AMPs) are the main components of the plant innate immune system. Defensins represent the most important AMP family involved in defense and non-defense functions. In this work, global RNA sequencing and de novo transcriptome assembly were performed to explore the diversity of defensin-like (DEFL) genes in the wheat Triticum kiharae and to study their role in induced resistance (IR) mediated by the elicitor metabolites of a non-pathogenic strain FS-94 of Fusarium sambucinum. Using a combination of two pipelines for DEFL mining in transcriptome data sets, as many as 143 DEFL genes were identified in T. kiharae, the vast majority of them represent novel genes. According to the number of cysteine residues and the cysteine motif, wheat DEFLs were classified into ten groups. Classical defensins with a characteristic 8-Cys motif assigned to group 1 DEFLs represent the most abundant group comprising 52 family members. DEFLs with a characteristic 4-Cys motif CX{3,5}CX{8,17}CX{4,6}C named group 4 DEFLs previously found only in legumes were discovered in wheat. Within DEFL groups, subgroups of similar sequences originated by duplication events were isolated. Variation among DEFLs within subgroups is due to amino acid substitutions and insertions/deletions of amino acid sequences. To identify IR-related DEFL genes, transcriptional changes in DEFL gene expression during elicitor-mediated IR were monitored. Transcriptional diversity of DEFL genes in wheat seedlings in response to the fungus Fusarium oxysporum, FS-94 elicitors, and the combination of both (elicitors + fungus) was demonstrated, with specific sets of up- and down-regulated DEFL genes. DEFL expression profiling allowed us to gain insight into the mode of action of the elicitors from F. sambucinum. We discovered that the elicitors up-regulated a set of 24 DEFL genes. After challenge inoculation with F. oxysporum, another set of 22 DEFLs showed enhanced expression in IR-displaying seedlings. These DEFLs, in concert with other defense molecules, are suggested to determine enhanced resistance of elicitor-pretreated wheat seedlings. In addition to providing a better understanding of the mode of action of the elicitors from FS-94 in controlling diseases, up-regulated IR-specific DEFL genes represent novel candidates for genetic transformation of plants and development of pathogen-resistant crops.

Cysteine-rich peptides (CRPs) play an important role in plant physiology. However, their role in resistance induced by biogenic elicitors remains poorly understood. Using whole-genome transcriptome sequencing and our CRP search algorithm, we analyzed the repertoire of CRPs in tomato Solanum lycopersicum L. in response to Fusarium oxysporum infection and elicitors from F. sambucinum. We revealed 106 putative CRP transcripts belonging to different families of antimicrobial peptides (AMPs), signaling peptides (RALFs), and peptides with non-defense functions (Major pollen allergen of Olea europaea (Ole e 1 and 6), Maternally Expressed Gene (MEG), Epidermal Patterning Factor (EPF)), as well as pathogenesis-related proteins of families 1 and 4 (PR-1 and 4). We discovered a novel type of 10-Cys-containing hevein-like AMPs named SlHev1, which was up-regulated both by infection and elicitors. Transcript profiling showed that F. oxysporum infection and F. sambucinum elicitors changed the expression levels of different overlapping sets of CRP genes, suggesting the diversification of functions in CRP families. We showed that non-specific lipid transfer proteins (nsLTPs) and snakins mostly contribute to the response of tomato plants to the infection and the elicitors. The involvement of CRPs with non-defense function in stress reactions was also demonstrated. The results obtained shed light on the mode of action of F. sambucinum elicitors and the role of CRP families in the immune response in tomato.

Keywords: hevein-like antimicrobial peptides; antifungal activity; antifungal determinants; synergy; chemosensitization; tebuconazole; plant pathogenic fungi.

Non-specific lipid-transfer proteins (nsLTPs) represent a family of plant antimicrobial peptides (AMPs) implicated in diverse physiological processes. However, their role in induced resistance (IR) triggered by non-pathogenic fungal strains and their metabolites is poorly understood. In this work, using RNA-seq data and our AMP search pipeline, we analyzed the repertoire of nsLTP genes in the wheat Triticum kiharae and studied their expression in response to Fusarium oxysporum infection and treatment with the intracellular metabolites of Fusarium sambucinum FS-94. A total of 243 putative nsLTPs were identified, which were classified into five structural types and characterized. Expression analysis showed that 121 TkLTPs including sets of paralogs with identical mature peptides displayed specific expression patters in response to different treatments pointing to their diverse roles in resistance development. We speculate that upregulated nsLTP genes are involved in protection due to their antimicrobial activity or signaling functions. Furthermore, we discovered that in IR-displaying plants, a vast majority of nsLTP genes were downregulated, suggesting their role as negative regulators of immune mechanisms activated by the FS-94 elicitors. The results obtained add to our knowledge of the role of nsLTPs in IR and provide candidate molecules for genetic engineering of crops to enhance disease resistance.

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