Bacteria of the genus Pseudomonas occupy diverse environments. The Pseudomonas fluorescens group is particularly well-known for its plant-beneficial properties including pathogen suppression. Recent observations that some strains of this group also cause lethal infections in insect larvae, however, point to a more versatile ecology of these bacteria. We show that 26 P. fluorescens group strains, isolated from three continents and covering three phylogenetically distinct sub-clades, exhibited different activities toward lepidopteran larvae, ranging from lethal to avirulent. All strains of sub-clade 1, which includes Pseudomonas chlororaphis and Pseudomonas protegens, were highly insecticidal regardless of their origin (animals, plants). Comparative genomics revealed that strains in this sub-clade possess specific traits allowing a switch between plant-and insect-associated lifestyles. We identified 90 genes unique to all highly insecticidal strains (sub-clade 1) and 117 genes common to all strains of sub-clade 1 and present in some moderately insecticidal strains of sub-clade 3. Mutational analysis of selected genes revealed the importance of chitinase C and phospholipase C in insect pathogenicity. The study provides insight into the genetic basis and phylogenetic distribution of traits defining insecticidal activity in plant-beneficial pseudomonads. Strains with potent dual activity against plant pathogens and herbivorous insects have great potential for use in integrated pest management for crops.

HighlightPlant elicitor peptides (Peps) co-evolved with their receptors, resulting in interfamily incompatibility of Pep recognition. In contrast, operation of defence pathways by Pep receptors is conserved within the flowering plants.

Particular groups of plant-beneficial fluorescent pseudomonads are not only root colonizers that provide plant disease suppression, but in addition are able to infect and kill insect larvae. The mechanisms by which the bacteria manage to infest this alternative host, to overcome its immune system, and to ultimately kill the insect are still largely unknown. However, the investigation of the few virulence factors discovered so far, points to a highly multifactorial nature of insecticidal activity. Antimicrobial compounds produced by fluorescent pseudomonads are effective weapons against a vast diversity of organisms such as fungi, oomycetes, nematodes, and protozoa. Here, we investigated whether these compounds also contribute to insecticidal activity. We tested mutants of the highly insecticidal strains Pseudomonas protegens CHA0, Pseudomonas chlororaphis PCL1391, and Pseudomonas sp. CMR12a, defective for individual or multiple antimicrobial compounds, for injectable and oral activity against lepidopteran insect larvae. Moreover, we studied expression of biosynthesis genes for these antimicrobial compounds for the first time in insects. Our survey revealed that hydrogen cyanide and different types of cyclic lipopeptides contribute to insecticidal activity. Hydrogen cyanide was essential to full virulence of CHA0 and PCL1391 directly injected into the hemolymph. The cyclic lipopeptide orfamide produced by CHA0 and CMR12a was mainly important in oral infections. Mutants of CMR12a and PCL1391 impaired in the production of the cyclic lipopeptides sessilin and clp1391, respectively, showed reduced virulence in injection and feeding experiments. Although virulence of mutants lacking one or several of the other antimicrobial compounds, i.e., 2,4-diacetylphloroglucinol, phenazines, pyrrolnitrin, or pyoluteorin, was not reduced, these metabolites might still play a role in an insect background since all investigated biosynthetic genes for antimicrobial compounds of strain CHA0 were expressed at some point during insect infection. In summary, our study identified new factors contributing to insecticidal activity and extends the diverse functions of antimicrobial compounds produced by fluorescent pseudomonads from the plant environment to the insect host.

The endogenous Arabidopsis (Arabidopsis thaliana) peptides, AtPeps, elicit an innate immune response reminiscent of patterntriggered immunity. Detection of various danger signals, including microbe-associated molecular patterns (MAMPs), leads to elevated transcription of PROPEPs, the AtPep precursors, and PEPRs, the AtPep receptors. It has been hypothesized that AtPeps are involved in enhancing pattern-triggered immunity. Following this idea, we analyzed the relationship between MAMP-and AtPep-elicited signaling. We found that the perception of MAMPs enhanced a subsequent AtPep-triggered production of reactive oxygen species (ROS). Intriguingly, other components of AtPep-triggered immunity like Ca 2+ influx, mitogen-activated protein kinase phosphorylation, ethylene production, and expression of early defense genes, as well as ROS-activated genes, remained unchanged. By contrast, treatment with methyl jasmonate promoted an increase of all analyzed AtPep-triggered responses. We positively correlated the intensities of generic AtPep-triggered responses with the abundance of the two AtPep receptors by generating constitutively expressing PEPR1 and PEPR2 transgenic lines and by analyzing pepr1 and pepr2 mutants. Further, we show that enhanced, as well as basal, ROS production triggered by AtPeps is absent in the double mutant of the respiratory burst oxidase homologs D and F (rbohD rbohF). We present evidence that the enhancement of AtPep-triggered ROS is not based on changes in the ROS detoxification machinery and is independent of mitogen-activated protein kinase and Ca 2+ signaling pathways. Taken together, these results indicate an additional level of regulation besides receptor abundance for the RbohD/RbohF-dependent production of AtPep-elicited ROS, which is specifically operated by MAMP-triggered pathways.

HighlightDysfunction of the Pep-PEPR system and its interplay with JA signalling results in increased plant susceptibility towards herbivore attack indicating that endogenous signalling also contributes to herbivore defence.