Plant phenolic volatiles inhibit quorum sensing in pectobacteria and reduce their virulence by potential binding to ExpI and ExpR proteins.

Quorum sensing (QS) is a population density-dependent regulatory system in bacteria that couples gene expression to cell density through accumulation of diffusible signaling molecules. Pectobacteria are causal agents of soft rot disease in a range of economically important crops. They rely on QS to coordinate their main virulence factor, production of plant cell wall degrading enzymes (PCWDEs). Plants have evolved an array of antimicrobial compounds to anticipate and cope with pathogens, of which essential oils (EOs) are widely recognized. Here, volatile EOs, carvacrol and eugenol, were shown to specifically interfere with QS, the master regulator of virulence in pectobacteria, resulting in strong inhibition of QS genes, biofilm formation and PCWDEs, thereby leading to impaired infection. Accumulation of the signal molecule N-acylhomoserine lactone declined upon treatment with EOs, suggesting direct interaction of EOs with either homoserine lactone synthase (ExpI) or with the regulatory protein (ExpR). Homology models of both proteins were constructed and docking simulations were performed to test the above hypotheses. The resulting binding modes and docking scores of carvacrol and eugenol support potential binding to ExpI/ExpR, with stronger interactions than previously known inhibitors of both proteins. The results demonstrate the potential involvement of phytochemicals in the control of Pectobacterium.

Expression levels of antimicrobial peptide tachyplesin I in transgenic Ornithogalum lines affect the resistance to Pectobacterium infection.

The genus Ornithogalum includes several ornamental species that suffer substantial losses from bacterial soft rot caused by Pectobacteria. The absence of effective control measures for use against soft rot bacteria led to the initiation of a project in which a small antimicrobial peptide from an Asian horseshoe crab, tachyplesin (tpnI), was introduced into two commercial cultivars: O. dubium and O. thyrsoides. Disease severity and bacterial colonization were examined in transgenic lines expressing this peptide. Disease resistance was evaluated in six lines of each species by measuring bacterial proliferation in the plant tissue. Three transgenic lines of each species were subjected to further analysis in which the expression level of the transgene was evaluated using RT-PCR and qRT-PCR. The development of disease symptoms and bacterial colonization of the plant tissue were also examined using GFP-expressing strain of P. carotovorum subsp. brasiliense Pcb3. Confocal-microscopy imaging revealed significantly reduced quantities of bacterial cells in the transgenic plant lines that had been challenged with the bacterium. The results clearly demonstrate that tpnI expression reduces bacterial proliferation, colonization and disease symptom (reduced by 95-100%) in the transgenic plant tissues. The quantity of tpnI transcripts, as measured by qRT-PCR, was negatively correlated with the protection afforded to the plants, as measured by the reduced severity of disease symptoms in the tissue.

Plant phenolic acids affect the virulence of Pectobacterium aroidearum and P. carotovorum ssp. brasiliense via quorum sensing regulation.

Several studies have reported effects of the plant phenolic acids cinnamic acid (CA) and salicylic acid (SA) on the virulence of soft rot enterobacteria. However, the mechanisms involved in these processes are not yet fully understood. Here, we investigated whether CA and SA interfere with the quorum sensing (QS) system of two Pectobacterium species, P. aroidearum and P. carotovorum ssp. brasiliense, which are known to produce N-acyl-homoserine lactone (AHL) QS signals. Our results clearly indicate that both phenolic compounds affect the QS machinery of the two species, consequently altering the expression of bacterial virulence factors. Although, in control treatments, the expression of QS-related genes increased over time, the exposure of bacteria to non-lethal concentrations of CA or SA inhibited the expression of QS genes, including expI, expR, PC1_1442 (luxR transcriptional regulator) and luxS (a component of the AI-2 system). Other virulence genes known to be regulated by the QS system, such as pecS, pel, peh and yheO, were also down-regulated relative to the control. In agreement with the low levels of expression of expI and expR, CA and SA also reduced the level of the AHL signal. The effects of CA and SA on AHL signalling were confirmed in compensation assays, in which exogenous application of N-(ß-ketocaproyl)-l-homoserine lactone (eAHL) led to the recovery of the reduction in virulence caused by the two phenolic acids. Collectively, the results of gene expression studies, bioluminescence assays, virulence assays and compensation assays with eAHL clearly support a mechanism by which CA and SA interfere with Pectobacterium virulence via the QS machinery.

Effects of plant antimicrobial phenolic compounds on virulence of the genus Pectobacterium.

Pectobacterium spp. are among the most devastating necrotrophs, attacking more than 50% of angiosperm plant orders. Their virulence strategy is based mainly on the secretion of exoenzymes that degrade the cell walls of their hosts, providing nutrients to the bacteria, but conversely, exposing the bacteria to plant defense compounds. In the present study, we screened plant-derived antimicrobial compounds, mainly phenolic acids and polyphenols, for their ability to affect virulence determinants including motility, biofilm formation and extracellular enzyme activities of different Pectobacteria: Pectobacterium carotovorum, P. brasiliensis, P. atrosepticum and P. aroidearum. In addition, virulence assays were performed on three different plant hosts following exposure of the bacteria to selected phenolic compounds. These experiments showed that cinnamic, coumaric, syringic and salicylic acids and catechol can considerably reduce disease severity, ranging from 20 to 100%. The reduced disease severity was not only the result of reduced bacterial growth, but also of a direct effect of the compounds on important bacterial virulence determinants, including pectolytic and proteolytic exoenzyme activities, that were reduced by 50-100%. This is the first report revealing a direct effect of phenolic compounds on virulence factors in a wide range of Pectobacterium strains.

Genetic transformation of Ornithogalum via particle bombardment and generation of Pectobacterium carotovorum-resistant plants.

Bacterial soft rot caused by Pectobacterium carotovorum subsp. carotovorum (Pcc) is one of the most devastating diseases of Ornithogalum species. No effective control measures are currently available to use against this pathogen; thus, introduction of resistant genes via genetic transformation into this crop is a promising approach. Tachyplesin I, an antimicrobial peptide, has been shown to effectively control numerous pathogenic bacteria, including Pcc. In this study, liquid-grown cell clusters of Ornithogalum dubium and Ornithogalum thyrsoides were bombarded with a pCAMBIA2301 vector containing a celI leader sequence fused to a gene encoding tachyplesin I, a neomycin phosphotransferase (nptII) gene that served as a selectable marker and a ß-glucuronidase (GUS) gene that served as a reporter. Selection was carried out in the dark in liquid medium containing 80mg/L kanamycin. Regeneration was executed in the light after 6-14 months depending on the cultivar. Hundreds of transgenic plantlets were produced and their identity was confirmed through GUS activity assays. PCR and RT-PCR were used to confirm the presence of the target, reporter and selection genes in the divergent lines of plantlets. The resistance of the O. dubium plants to Pcc was evaluated in vitro, following infection with a highly virulent isolate from calla lily. Although control plantlets were completely macerated within a week, 87 putative transgenic subclones displayed varying levels of disease resistance. During three growing seasons in the greenhouse, the transgenic O. dubium lines grew poorly, whereas the transgenic O. thyrsoides plants grew similarly to non-transgenic plants.

A systemic response of geophytes is demonstrated by patterns of protein expression and the accumulation of signal molecules in Zantedeschia aethiopica.

In geophyte plants, such as Zantedeschia, individual leaves are directly connected to a specialized underground storage organ (rhizome/tuber), raising a question regarding systemic resistance as a mechanism of defense. A systemic response requires a transfer of a signal through the storage organ which has been evolutionary adapted to store food, minerals and moisture for seasonal growth and development. We have characterized the nature of induced defense responses in Zantedeschia aethiopica, a rhizomatous (tuber-like) ornamental plant by the application of local elicitation using two well-known defense elicitors, benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) and methyl jasmonate (MJ). The system consisted leaves in which local responses were directly induced, and systemically responsive leaves in which defense molecules were detected, demonstrating a transported vascular signal. Using anatomical and biochemical tools and local elicitation with MJ, the systemic nature of the response was verified in adjacent leaves by unique protein expression patterns; similarly polyphenol oxidase (PPO) activity was found to increase systemically in all parts of the locally induced plants, including the rhizome, and adjacent leaves; finally, significant accumulation of defense signal molecules such as salicylic and jasmonic acids was recorded in local and systemic leaves following elicitation with BTH. Anatomical sections through the leaves and the rhizome revealed that to be transferred from one leaf to its neighbor, signal molecules must have been transferred through the storage organ. The collected data strongly support our hypothesis that defense signals may and are transferred through the storage organ in monocot geophytes.