plipastatin-a1 and fengycin

Non-self-recognition of microorganisms partly relies on the perception of microbe-associated molecular patterns (MAMPs) and leads to the activation of an innate immune response. Bacillus subtilis produces three main families of cyclic lipopeptides (LPs), namely surfactins, iturins and fengycins. Although LPs are involved in induced systemic resistance (ISR) activation, little is known about defence responses induced by these molecules and their involvement in local resistance to fungi. Here, we showed that purified surfactin, mycosubtilin (iturin family) and plipastatin (fengycin family) are perceived by grapevine plant cells. Although surfactin and mycosubtilin stimulated grapevine innate immune responses, they differentially activated early signalling pathways and defence gene expression. By contrast, plipastatin perception by grapevine cells only resulted in early signalling activation. Gene expression analysis suggested that mycosubtilin activated salicylic acid (SA) and jasmonic acid (JA) signalling pathways, whereas surfactin mainly induced an SA-regulated response. Although mycosubtilin and plipastatin displayed direct antifungal activity, only surfactin and mycosubtilin treatments resulted in a local long-lasting enhanced tolerance to the necrotrophic fungus Botrytis cinerea in grapevine leaves. Moreover, challenge with specific strains overproducing surfactin and mycosubtilin led to a slightly enhanced stimulation of the defence response compared with the LP-non-producing strain of B. subtilis. Altogether, our results provide the first comprehensive view of the involvement of LPs from B. subtilis in grapevine plant defence and local resistance against the necrotrophic pathogen Bo. cinerea. Moreover, this work is the first to highlight the ability of mycosubtilin to trigger an immune response in plants.

Topics: Bacillus subtilis; Botrytis; Fatty Acids; Lipopeptides; Oligopeptides; Peptides, Cyclic; Vitis

Bacillus strains produce non-ribosomal lipopeptides that can be grouped into three families: surfactins or lichenysins, iturins and fengycins or plispastatins. These biosurfactants show a broad spectrum of biological activities. To detect strains able to produce these lipopeptides, a new polymerase chain reaction screening approach was developed using degenerated primers based on the intraoperon alignment of adenylation and thiolation nucleic acid domains of all enzymes implicated in the biosynthesis of each lipopeptide family. The comparative bioinformatics analyses of each operon led to the design of four primer pairs for the three families taking into account the differences between open reading frames of each synthetase gene. Tested on different Bacillus sp. strains, this technique was used successfully to detect not only the expected genes in the lipopeptide producing strains but also the presence of a plispastatin gene in Bacillus subtilis ATCC 21332 and a gene showing a high similarity with the polyketide synthase type I gene in the B. subtilis ATCC 6633 genome. It also led to the discovery of the presence of non-ribosomal peptide synthetase genes in Bacillus thuringiensis serovar berliner 1915 and in Bacillus cereus LMG 2098. In addition, this work highlighted the differences between the fengycin and plipastatin operon at DNA level.

Topics: Bacillus; Bacillus cereus; Bacillus subtilis; Bacillus thuringiensis; Bacterial Proteins; Cloning, Molecular; DNA Primers; DNA, Bacterial; Fatty Acids; Genes, Bacterial; Lipopeptides; Oligopeptides; Open Reading Frames; Operon; Peptide Biosynthesis, Nucleic Acid-Independent; Peptide Synthases; Peptides, Cyclic; Polyketide Synthases; Polymerase Chain Reaction