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Numerous functions in pathogenic Pectobacterium are regulated by quorum sensing (QS). Two different aiiA genes isolated from Bacillus sp. A24(aiiA A24 ) and Bacillus sp. DMS133(aiiA DMS133 ) were used. Both genes encode acyl-homoserine lactonase (AiiA), which disrupts QS in Pectobacterium. To investigate the effect of different AiiAs on the inhibition of Pectobacterium carotovorum pathogenicity, two aiiA genes from different Bacillus strains were cloned and the resulting plasmids pME6863 (aiiA A24 ) and pME7080 (aiiA DMS133 ) were transformed into P. carotovorum EMPCC cells. The effects of different lactonases on virulence features such as enzymatic activity, twitching and swimming motilities, and production of pellicle and biofilm formation were investigated. In EMPCC/pME6863, twitching and swimming motilities, and pellicle production were significantly reduced compared with EMPCC/pME7080. Quantitative real-time PCR (qRT-PCR) was used to measure virulence gene expression in transformed cells compared with expression levels in wild-type EMPCC. The expression of peh and hrpL genes was greatly reduced in EMPCC/pME6863 compared with EMPCC/pME7080. The sequence alignment and molecular dynamic modeling of two different AiiA A24 and AiiA DMS133 proteins suggested that the replacement of proline 210 from AiiA A24 to serine in AiiA DMS133 caused the reduction of enzyme activity in AiiA DMS133 ., (© 2024. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

Potatoes (Solanum tuberosum L.) are one of the world's major staple crops. In stored potatoes, Pectobacterium carotovorum subsp carotovorum causes soft rot. As a result of the rapid spread of the disease during post-harvest storage, potato production suffers huge losses. By detecting disease early and controlling it promptly, losses can be minimized. The profile of volatiles of plants can be altered by phytopathogens. Identifying unique volatile organic compounds (VOCs) as biomarkers for early disease detection has attracted considerable research attention. This study compared the VOC profiles of healthy and soft rot inoculated potatoes (cv. "Kufri Pukhraj") over a time course using gas chromatography-mass spectrometry (GC-MS). It was found that there was a differential emission of 27 VOCs between healthy non-inoculated potatoes and soft rot inoculated potatoes. Among 27 VOCs, only five (1-octen-3-ol, 2-methylisoborneol, 3-octanone, 1,4-dimethyladamantane, and 2-methyl-2-bornene) were found exclusively in soft rot inoculated potatoes, suggesting them potential biomarker for non-destructive prediction of soft rot disease in potatoes. Reactive oxygen species (H 2 O 2 ) and phytohormone methyl-jasmonate (MeJa) levels increased transiently on infection with soft rot. The analysis of the primary metabolism of soft rot infected tubers at three different stages suggests metabolic reprogramming that occurs at the early stage of infection, possibly leading to biomarker volatile emission. Based on these results, it appears that the initial potato-soft rot bacteria interaction initiates metabolic reprogramming mainly through H 2 O 2 and the MeJa signalling pathway. In asymptomatic potatoes, these biomarkers may be promising candidates for non-destructive detection of soft rot at an early stage. These biomarkers can be used to develop an e-nose sensor to predict soft rot in the future., (© 2024 Scandinavian Plant Physiology Society.)

Based on Single moleculereal time(SMRT)sequencing technology, the high-quality whole genome sequence of Pectobacterium carotovorum (PC1) was obtained by the PacBio RS II sequencer. The genome is a single circular chromosome of 5.3 Mb in size, containing three kinds of m6A methylation modification by SMRT Portal analysis. Genome annotation showed that 575 virulence factor genes, 304 drug resistance genes, 774 pathogen genes, 7 secretory systems and 22 pairs of two-component regulatory system could be relevant to bacterial pathogenicity. In addition, the average nucleotide identities (ANI) analysisshowed that the PC1 exhibited the highest homology with the Pectobacteriumcarotovorumsubsp.carotovorumstrain BP201601.1 (NZ_CP034236). There are 28 unique gene families to PC1 using cluster analysis of gene families. According to the analysis of key pathogenic genes, we have obtained three kinds of highly conserved genes related to cell wall degrading enzymes, including 19 pectinase genes, 25 cellulase genes and 22 protease genes. Our studies have provided a theoretical basis for investigation of bacterial soft rot and biological specific bactercides of PC1., (Copyright © 2021. Published by Elsevier Inc.)

Managed aquifer recharge (MAR) can provide irrigation water and overcome water scarcity in agriculture. Removal of potentially present plant pathogens during MAR is essential to prevent crop diseases. We studied the die-off of three plant pathogenic bacteria in water microcosms with natural or filtered tile drainage water (TDW) at 10 and 25°C and with natural anoxic aquifer water (AW) at 10°C from a MAR site. These bacteria were: Ralstonia solanacearum (bacterial wilt), and the soft rot Pectobacteriaceae (SRP) Dickeya solani and Pectobacterium carotovorum sp. carotovorum (soft rot, blackleg). They are present in surface waters and cause destructive crop diseases worldwide which have been linked to contaminated irrigation water. Nevertheless, little is known about the survival of the SRP in aqueous environments and no study has investigated the persistence of R. solanacearum under natural anoxic conditions. We found that all bacteria were undetectable in 0.1 mL samples within 19 days under oxic conditions in natural TDW at 10°C, using viable cell counting, corresponding to 3-log10 reduction by die-off. The SRP were no longer detected within 6 days at 25°C, whereas R. solanacearum was detectable for 25 days. Whereas in anoxic natural aquifer water at 10°C, the bacterial concentrations declined slower and the detection limit was reached within 56 days. Finally, we modelled the inactivation curves with a modified Weibull model that can simulate different curve shapes such as shoulder phenomena in the beginning and long tails reflecting persistent bacterial populations. The non-linear model was shown to be a reliable tool to predict the die-off of the analysed plant pathogenic bacteria, suggesting its further application to other pathogenic microorganisms in the context of microbial risk assessment., Competing Interests: The authors declare no competing interests in general and with any of the commercial affiliations. This commercial affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials.

In spite of the positive effects of bacteria on health, certain species are harmful, and therefore, animals must weigh nutritional benefits against negative post-ingestion consequences and adapt their behavior accordingly. Here, we use Drosophila to unravel how the immune system communicates with the brain, enabling avoidance of harmful foods. Using two different known fly pathogens, mildly pathogenic Erwinia carotovora (Ecc15) and highly virulent Pseudomonas entomophila (Pe), we analyzed preference behavior in naive flies and after ingestion of either of these pathogens. Although survival assays confirmed the harmful effect of pathogen ingestion, naive flies preferred the odor of either pathogen to air and also to harmless mutant bacteria, suggesting that flies are not innately repelled by these microbes. By contrast, feeding assays showed that, when given a choice between pathogenic and harmless bacteria, flies-after an initial period of indifference-shifted to a preference for the harmless strain, a behavior that lasted for several hours. Flies lacking synaptic output of the mushroom body (MB), the fly's brain center for associative memory formation, lost the ability to distinguish between pathogenic and harmless bacteria, suggesting this to be an adaptive behavior. Interestingly, this behavior relied on the immune receptors PGRP-LC and -LE and their presence in octopaminergic neurons. We postulate a model wherein pathogen ingestion triggers PGRP signaling in octopaminergic neurons, which in turn relay the information about the harmful food source directly or indirectly to the MB, where an appropriate behavioral output is generated., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Given the major threat of phytopathogenic bacteria to food production and ecosystem stability worldwide, novel alternatives to conventional chemicals-based agricultural practices are needed to combat these bacteria. The objective of this study is to evaluate the ability of Pseudomonas segetis strain P6, which was isolated from the Salicornia europaea rhizosphere, to act as a potential biocontrol agent given its plant growth-promoting (PGP) and quorum quenching (QQ) activities. Seed biopriming and in vivo assays of tomato plants inoculated with strain P6 resulted in an increase in seedling height and weight. We detected QQ activity, involving enzymatic degradation of signal molecules in quorum sensing communication systems, against a broad range of N-acylhomoserine lactones (AHLs). HPLC-MRM data and phylogenetic analysis indicated that the QQ enzyme was an acylase. The QQ activity of strain P6 reduced soft rot symptoms caused by Dickeya solani, Pectobacterium atrosepticum and P. carotovorum on potato and carrot. In vivo assays showed that the PGP and QQ activities of strain P6 protect tomato plants against Pseudomonas syringae pv. tomato, indicating that strain P6 could have biotechnological applications. To our knowledge, this is the first report to show PGP and QQ activities in an indigenous Pseudomonas strain from Salicornia plants.

Quorum-quenching (QQ) enzymes can block the quorum-sensing (QS) system and prevent the expression of QS-controlled pathogenic factors in bacteria. However, the low expression levels of QQ proteins in the original host bacteria have affected their widespread application. In this study, we heterologously expressed momL, encoding a QQ enzyme with high activity, in Lysobacter enzymogenes. A "yellow-to-white" selection marker and the high-constitutive-expression promoter P groEL were used in this novel heterologous expression system. In addition, we optimized the spacer between the SD sequence and the initiator to improve the efficiency of the expression system by 1.54-fold. The engineered strain LeMomL degraded the AHL molecule and the virulence factors of Pectobacterium carotovorum subsp. carotovora (Pcc). Additionally, LeMomL significantly decreased the disease caused by Pcc in Chinese cabbages and carrot root tissues. In conclusion, this novel and facile L. enzymogenes expression strategy has good prospects and is an ideal approach for foreign protein expression.

Surviving infection requires immune and repair mechanisms. Developing organisms face the additional challenge of integrating these mechanisms with tightly controlled developmental processes. The larval Drosophila midgut lacks dedicated intestinal stem cells. We show that, upon infection, larvae perform limited repair using adult midgut precursors (AMPs). AMPs differentiate in response to damage to generate new enterocytes, transiently depleting their pool. Developmental delay allows for AMP reconstitution, ensuring the completion of metamorphosis. Notch signaling is required for the differentiation of AMPs into the encasing, niche-like peripheral cells (PCs), but not to differentiate PCs into enterocytes. Dpp (TGF-β) signaling is sufficient, but not necessary, to induce PC differentiation into enterocytes. Infection-induced JAK-STAT pathway is both required and sufficient for differentiation of AMPs and PCs into new enterocytes. Altogether, this work highlights the constraints imposed by development on an organism's response to infection and demonstrates the transient use of adult precursors for tissue repair., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Black rot caused by Xanthomonas campestris pv. campestris (Xcc) is the most damaging disease in Brassica crops around the world. In this study, we developed a molecular marker specific to Xcc race 5. To do this, the available whole genome sequences of Xcc races/strains and Xc subspecies were aligned and identified a highly variable genomic region (XccR5-89.2). Subsequently, a primer set covering the 'XccR5-89.2' region was designed and tested against the genomic DNA of Xcc races/strains, Xc subspecies and other plant-infecting bacterial strains ( Pseudomonas syringae pv. maculicola and Erwinia carotovora subsp. carotovora ). The results showed that the 'XccR5-89.2' primer pair amplified a 2,172-bp fragment specific to Xcc race 5. Moreover, they also amplified a 1,515-bp fragment for Xcc race 1 and an over 3,000-bp fragment for Xcc race 3. However, they did not amplify any fragments from the remaining Xcc races/strains, subspecies or other bacterial strains. The 'XccR5-89.2' primer pair was further PCR amplified from race-unknown Xcc strains and ICMP8 was identified as race 5 among nine race-unknown Xcc strains. Further cloning and sequencing of the bands amplified from race 5 and ICMP8 with 'XccR5-89.2' primers revealed both carrying identical sequences. The results showed that the 'XccR5-89.2' marker can effectively and proficiently detect, and identify Xcc race 5 from Xcc races/strains, subspecies and other plant-infecting bacteria. To our knowledge, this is the first report for an Xcc race 5-specific molecular marker.

MomL is a marine-derived quorum-quenching (QQ) lactonase which can degrade various N -acyl homoserine lactones (AHLs). Intentional modification of MomL may lead to a highly efficient QQ enzyme with broad application potential. In this study, we used a rapid and efficient method combining error-prone polymerase chain reaction (epPCR), high-throughput screening and site-directed mutagenesis to identify highly active MomL mutants. In this way, we obtained two candidate mutants, MomL I144V and MomL V149A . These two mutants exhibited enhanced activities and blocked the production of pathogenic factors of Pectobacterium carotovorum subsp. carotovorum (Pcc) . Besides, seven amino acids which are vital for MomL enzyme activity were identified. Substitutions of these amino acids (E238G/K205E/L254R) in MomL led to almost complete loss of its QQ activity. We then tested the effect of MomL and its mutants on Pcc -infected Chinese cabbage. The results indicated that MomL and its mutants (MomL L254R , MomL I144V , MomL V149A ) significantly decreased the pathogenicity of Pcc . This study provides an efficient method for QQ enzyme modification and gives us new clues for further investigation on the catalytic mechanism of QQ lactonase.

Many Gram-negative aquacultural and agricultural pathogens control virulence factor expression through a quorum-sensing (QS) mechanism involving the production of N-acylhomoserine (AHL) signalling molecules. Thus, the interruption of QS systems by the enzymatic degradation of signalling molecules, known as quorum quenching (QQ), has been proposed as a novel strategy to combat these infections. Given that the symbiotic bacteria of marine invertebrates are considered to be an important source of new bioactive molecules, this study explores the presence of AHL-degrading bacteria among 827 strains previously isolated from the microbiota of anemones and holothurians. Four of these strains (M3-1, M1-14, M3-13 and M9-54-2), belonging to the species Stenotrophomonas maltophilia, were selected on the basis of their ability to degrade a broad range of AHLs, and the enzymes involved in their activity were identified. Strain M9-54-2, which showed the strongest AHL-degrading activity, was selected for further study. High-performance liquid chromatography-mass-spectrometry confirmed that the QQ enzyme is not a lactonase. Strain M9-54-2 degraded AHL accumulation and reduced the production of enzymatic activity in Pectobacterium carotovorum CECT 225 T and Vibrio coralliilyticus VibC-Oc-193 in in vitro co-cultivation experiments. The effect of AHL inactivation was confirmed by a reduction in potato tuber maceration and brine shrimp (Artemia salina) mortality caused by P. carotovorum and Vibrio coralliilyticus, respectively. This study strengthens the evidence of marine organisms as an underexplored and promising source of QQ enzymes, useful to prevent infections in aquaculture and agriculture. To our knowledge, this is the first time that anemones and holothurians have been studied for this purpose.

Solanum tuberosum, commonly known as the potato, is a worldwide food staple. During harvest, storage, and distribution the crop is at risk of mechanical damage. Wounding of the tuber skin can also become a point of entry for bacterial and fungal pathogens, resulting in substantial agricultural losses. Building on the proposal that potato tubers produce metabolites to defend against microbial infection during early stages of wound healing before protective suberized periderm tissues have developed, we assessed extracts of wound tissues from four potato cultivars with differing skin morphologies (Norkotah Russet, Atlantic, Chipeta, and Yukon Gold). These assays were conducted at 0, 1, 2, 3 and 7 days post wounding against the plant pathogen Erwinia carotovora and a non-pathogenic Escherichia coli strain that served as a control. For each of the potato cultivars, only polar wound tissue extracts demonstrated antibacterial activity. The polar extracts from earlier wound-healing time points (days 0, 1 and 2) displayed notably higher antibacterial activity against both strains than the later wound-healing stages (days 3 and 7). These results support a burst of antibacterial activity at early time points. Parallel metabolite profiling of the extracts revealed differences in chemical composition at different wound-healing time points and allowed for identification of potential marker compounds according to healing stage for each of the cultivars. It was possible to monitor the transformations in the metabolite profiles that could account for the phenomenon of temporal resistance by looking at the relative quantities of various metabolite classes as a function of time., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Thionins are a family of antimicrobial peptides. We performed in silico expression analyses of the 44 rice (Oryza sativa) thionins (OsTHIONs). Modulated expression levels of OsTHIONs under different treatments suggest their involvement in many processes, including biotic, abiotic, and nutritional stress responses, and in hormone signaling. OsTHION15 (LOC_Os06g32600) was selected for further characterization based on several in silico analyses. OsTHION15 in O. sativa subsp. indica 'KDML 105' was expressed in all of the tissues and organs examined, including germinating seed, leaves, and roots of seedlings and mature plants, and inflorescences. To investigate the antimicrobial activity of OsTHION15, we produced a recombinant peptide in Escherichia coli Rosetta-gami (DE3). The recombinant OsTHION15 exhibited inhibitory activities toward rice-pathogenic bacteria such as Xanthomonas oryzae pv. oryzae and Pectobacterium carotovorum pv. atroseptica, with minimum inhibitory concentrations of 112.6 and 14.1 µg ml -1 , respectively. A significant hyphal growth inhibition was also observed toward Fusarium oxysporum f. sp. cubense and Helminthosporium oryzae. In addition, we demonstrated the in planta antibacterial activity of this peptide in Nicotiana benthamiana against X. campestris pv. glycines. These activities suggest the possible application of OsTHION15 in plant disease control.

Four hundred and fifty bacteria were evaluated for antagonistic activity against bacterial soft rot of potato caused by Pectobacterium carotovorum sp strain II16. A strain Ar10 exhibiting potent antagonist activity has been identified as Bacillus amyloliquefaciens on the basis of biochemical and molecular characterization. Cell free supernatant showed a broad spectrum of antibacterial activity against human and phytopathogenic bacteria in the range of 10-60 AU/mL. Incubation of P. carotovorum cells with increasing concentrations of the antibacterial compound showed a killing rate of 94.8 and 96% at MIC and 2xMIC respectively. In addition, the antibacterial agent did not exert haemolytic activity at the active concentration and has been preliminary characterized by TLC and GC-MS as a glycolipid compound. Treatment of potato tubers with strain Ar10 for 72 h significantly reduced the severity of disease symptoms (100 and 85.05% reduction of necrosis deep / area and weight loss respectively). The same levels in disease symptoms severity was also recorded following treatment of potato tubers with cell free supernatant for 1 h. Data suggest that protection against potato soft rot disease may be related to glycolipid production by strain Ar10. The present study affords new alternatives for anti-Pectobacterium carotovorum bioactive compounds against the soft rot disease of potato., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Hfq is a RNA chaperone and participates in a wide range of cellular processes and pathways. In this study, mutation of hfq gene from Pectobacterium carotovorum subsp. carotovorum PccS1 led to significantly reduced virulence and plant cell wall-degrading enzyme (PCWDE) activities. In addition, the mutant exhibited decreased biofilm formation and motility and greatly attenuated carbapenem production as well as secretion of hemolysin coregulated protein (Hcp) as compared with wild-type strain PccS1. Moreover, a higher level of callose deposition was induced in Nicotiana benthamiana leaves when infiltrated with the mutant. A total of 26 small (s)RNA deletion mutants were obtained among a predicted 27 sRNAs, and three mutants exhibited reduced virulence in the host plant. These results suggest that hfq plays a key role in Pectobacterium virulence by positively impacting PCWDE production, secretion of the type VI secretion system, bacterial competition, and suppression of host plant responses.

The digestive tract is the first organ affected by the ingestion of foodborne bacteria. While commensal bacteria become resident, opportunistic or virulent bacteria are eliminated from the gut by the local innate immune system. Here we characterize a new mechanism of defense, independent of the immune system, in Drosophila melanogaster. We observed strong contractions of longitudinal visceral muscle fibers for the first 2 hours following bacterial ingestion. We showed that these visceral muscle contractions are induced by immune reactive oxygen species (ROS) that accumulate in the lumen and depend on the ROS-sensing TRPA1 receptor. We then demonstrate that both ROS and TRPA1 are required in a subset of anterior enteroendocrine cells for the release of the DH31 neuropeptide which activates its receptor in the neighboring visceral muscles. The resulting contractions of the visceral muscles favors quick expulsion of the bacteria, limiting their presence in the gut. Our results unveil a precocious mechanism of defense against ingested opportunistic bacteria, whether they are Gram-positive like Bacillus thuringiensis or Gram-negative like Erwinia carotovora carotovora. Finally, we found that the human homolog of DH31, CGRP, has a conserved function in Drosophila., Competing Interests: The authors have declared that no competing interests exist.

Vitamin B6 (VitB6) is an essential cofactor for >140 biochemical reactions. Also, VitB6 is a potent antioxidant and helps plants cope with both biotic and abiotic stress conditions. However, the role of VitB6 in plant disease resistance has yet to be confirmed using molecular biology approaches. Here, we analyzed the expression patterns of VitB6 biosynthetic genes, including the de novo (PDX1 [PDX1.2 and 1.3] and PDX2) and the salvage (SOS4) pathways during the response to Erwinia carotovora subsp. carotovora. By quantitative PCR, we found that the most significant upregulation in the transcript profile of PDX2, which showed a 9.2-fold increase in expression at 12 h post inoculation (hpi) compared to 24-48 hpi. We also detected significant upregulation of PDX1.2 and PDX1.3, which were 6.6- and 4.3-fold upregulated at 24 hpi compared to 12 hpi, while SOS4 showed only low-level expression. Also, at 24 hpi, a significant increase in superoxide dismutase, catalase, peroxidase, and polyphenol oxidase activities was observed in plants. Our findings confirm that the expression of de novo and salvage pathway genes is induced by E. carotovora and that this plays an important role in the regulation of defense response by modulating cellular antioxidant capacity., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Aims: Isolation and characterization of pectolytic bacteria associated with soft rot disease of potatoes in Nakuru, Kenya, to provide the basis for the development of disease control measures., Methods and Results: Potato tubers showing symptoms of soft rot were collected from different farms in Molo and Mau Narok regions within Nakuru county. Isolation was done using crystal violet pectate medium (CVPM). Out of the 71 isolates that showed growth on CVPM, pathogenicity tests revealed that 36 of them had the ability to macerate tissues of potato tubers. All the isolates yielded a fragment of approximately 1500 bp after 16S rDNA amplification. Using the BIOLOG microbial identification system, 20 bacterial isolates were identified as Pectobacterium carotovorum subsp. carotovorum, 7 were Pseudomonas fluorescens B while 9 were Ps. fluorescens A. Y1/Y2 primers successfully amplified pectate lyase-encoding (pel) gene, approximately 434 bp, in all the 20 P. carotovorum species. The virulence of the isolated strains to cause disease, according to pectinolytic tests, varied with change in incubation temperature of the test samples. Pectobacterium carotovorum strains were the most virulent at 30°C while disease severity due to infection by Ps. fluorescens A strains was high at 20°C compared to the other isolates., Conclusion: This study reveals the identity of pectolytic bacterial species from two genera, Pectobacterium and Pseudomonas, as causative agents of potato soft rot in Nakuru, Kenya., Significance and Impact of the Study: Research findings from this study will aid in developing suitable risk mitigation methods for adoption by farmers to prevent losses due to soft rot., (© 2018 The Society for Applied Microbiology.)

DUOX, a member of the NADPH oxidase family, acts as the first line of defense against enteric pathogens by producing microbicidal reactive oxygen species. DUOX is activated upon enteric infection, but the mechanisms regulating DUOX activity remain incompletely understood. Using Drosophila genetic tools, we show that enteric infection results in "pro-catabolic" signaling that initiates metabolic reprogramming of enterocytes toward lipid catabolism, which ultimately governs DUOX homeostasis. Infection induces signaling cascades involving TRAF3 and kinases AMPK and WTS, which regulate TOR kinase to control the balance of lipogenesis versus lipolysis. Enhancing lipogenesis blocks DUOX activity, whereas stimulating lipolysis via ATG1-dependent lipophagy is required for DUOX activation. Drosophila with altered activity in TRAF3-AMPK/WTS-ATG1 pathway components exhibit abolished infection-induced lipolysis, reduced DUOX activation, and enhanced susceptibility to enteric infection. Thus, this work uncovers signaling cascades governing inflammation-induced metabolic reprogramming and provides insight into the pathophysiology of immune-metabolic interactions in the microbe-laden gut epithelia., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Gut immunity is regulated by intricate and dynamic mechanisms to ensure homeostasis despite a constantly changing microbial environment. Several regulatory factors have been described to participate in feedback responses to prevent aberrant immune activity. Little is, however, known about how transcriptional programs are directly tuned to efficiently adapt host gut tissues to the current microbiome. Here we show that the POU/Oct gene nubbin (nub) encodes two transcription factor isoforms, Nub-PB and Nub-PD, which antagonistically regulate immune gene expression in Drosophila. Global transcriptional profiling of adult flies overexpressing Nub-PB in immunocompetent tissues revealed that this form is a strong transcriptional activator of a large set of immune genes. Further genetic analyses showed that Nub-PB is sufficient to drive expression both independently and in conjunction with nuclear factor kappa B (NF-κB), JNK and JAK/STAT pathways. Similar overexpression of Nub-PD did, conversely, repress expression of the same targets. Strikingly, isoform co-overexpression normalized immune gene transcription, suggesting antagonistic activities. RNAi-mediated knockdown of individual nub transcripts in enterocytes confirmed antagonistic regulation by the two isoforms and that both are necessary for normal immune gene transcription in the midgut. Furthermore, enterocyte-specific Nub-PB expression levels had a strong impact on gut bacterial load as well as host lifespan. Overexpression of Nub-PB enhanced bacterial clearance of ingested Erwinia carotovora carotovora 15. Nevertheless, flies quickly succumbed to the infection, suggesting a deleterious immune response. In line with this, prolonged overexpression promoted a proinflammatory signature in the gut with induction of JNK and JAK/STAT pathways, increased apoptosis and stem cell proliferation. These findings highlight a novel regulatory mechanism of host-microbe interactions mediated by antagonistic transcription factor isoforms.

Chlorogenic acid (CGA) plays an important role in protecting plants against pathogens and promoting human health. Although CGA accumulates to high levels in potato tubers, the key enzyme p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H) for CGA biosynthesis has not been isolated and functionally characterized in potato. In this work, we cloned StC3'H from potato and showed that it catalyzed the formation of caffeoylshikimate and CGA (caffeoylquinate) from p-coumaroyl shikimate and p-coumaroyl quinate, respectively, but was inactive towards p-coumaric acid in in vitro enzyme assays. When the expression of StC3'H proteins was blocked through antisense (AS) inhibition under the control of a tuber-specific patatin promoter, moderate changes in tuber yield as well as phenolic metabolites in the core tuber tissue were observed for several AS lines. On the other hand, the AS and control potato lines exhibited similar responses to a bacterial pathogen Pectobacterium carotovorum. These results suggest that StC3'H is implicated in phenolic metabolism in potato. They also suggest that CGA accumulation in the core tissue of potato tubers is an intricately controlled process and that additional C3'H activity may also be involved in CGA biosynthesis in potato., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The identification of phytopathogen proteins that are differentially expressed during the course of the establishment of an infection is important to better understand the infection process. In vitro approaches, using plant extracts added to culture medium, have been used to identify such proteins, but the biological relevance of these findings for in planta infection are often uncertain until confirmed by in vivo studies. Here, we compared the proteins of Pectobacterium carotovorum ssp. carotovorum strain PccS1 differentially expressed in Luria-Bertani medium supplemented with extracts of the ornamental plant Zantedeschia elliotiana cultivar 'Black Magic' (in vitro) and in plant tissues (in vivo) by two-dimensional electrophoresis coupled with mass spectrometry. A total of 53 differentially expressed proteins (>1.5-fold) were identified (up-regulated or down-regulated in vitro, in vivo or both). Proteins that exhibited increased expression in vivo but not in vitro, or in both conditions, were identified, and deletions were made in a number of genes encoding these proteins, four of which (clpP, mreB, flgK and eda) led to a loss of virulence on Z. elliotiana, although clpP and mreB were later also shown to be reduced in growth in rich and minimal media. Although clpP, flgK and mreB have previously been reported as playing a role in virulence in plants, this is the first report of such a role for eda, which encodes 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, a key enzyme in Entner-Doudoroff metabolism. The results highlight the value of undertaking in vivo as well as in vitro approaches for the identification of new bacterial virulence factors., (© 2016 BSPP AND JOHN WILEY & SONS LTD.)

Pluripotent stem cell activity is essential to maintain regeneration and homeostasis in the Drosophila midgut following environmental challenges. Although multiple pathways have been implicated in epithelial renewal, the underlying regulatory mechanisms and correlations between relevant genes and pathways remain elusive. In this study, we show that the zinc finger protein CG12744 plays an important role in the differentiation and regeneration of epithelial cells in response to oral infection with Erwinia carotovora carotovora 15. Knocking down CG12744 in enteroblasts decreased the post-infection proportion of enteroblasts and enterocytes and increased the post-infection number of enteroendocrine cells. In addition, in precursors, CG12744 affected the Osa, jun-N-terminal kinase and bone morphogenetic protein signaling pathways to control enterocyte differentiation. Finally, CG12744 maintained epithelial architecture and cell fate in enterocytes following an acute infectious challenge., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Three bacterial isolates were isolated from infected potato tubers showing soft and brown rots like symptoms as well as one isolate from infected peach tree showing crown gall symptom. The morphological, biochemical and molecular assays proved that bacterial isolates belonging to Pectobacterium carotovorum subsp. carotovorum, Ralstonia solanacearum, Dickeya spp. and Agrobacterium tumefaciens. The acetone (AcE) and n-butanol (ButE) extracts of Callistemon viminalis flowers and essential oil from aerial parts of Conyza dioscoridis as well as ButE of Eucalyptus camaldulensis bark are evaluated at different concentrations against the growth of the isolated bacteria. The diameter of inhibition zone (IZ) and the minimum inhibitory concentrations (MICs) are compared. Results indicated that the highest IZ values were 20.0 mm and 18.3 mm for E. camaldulensis bark ButE and C. viminalis flower ButE, respectively, against P. carotovorum; 16.3 mm and 16.0 mm for E. camaldulensis bark ButE and C. viminalis flower ButE, respectively, against R. solanacearum; 18.5 mm for C. viminalis flower AcE and C. dioscoridis aerial parts EO against Dickeya spp.; and 15.0 mm for C. viminalis flower AcE against A. tumefaciens. MICs ranged from <16 μg/mL for D. solani to >4000 μg/mL for A. tumefaciens. It was proved that C. viminalis flowers AcE contains mainly 5-hydroxymethylfurfural (20.6%), palmitic acid (18.5%), and pyrogallol (16.4%); while C. viminalis flower ButE contains palmitic acid (36.3%), 2-hydroxymyristic acid (9.4%), 5-hydroxymethylfurfural (7.2%), and shikimic acid (6.6%); whereas E. camaldulensis bark ButE contains 8-nonynoic acid methyl ester (45.6), camphor (30.9%), menthol (8.8%), and 1,8-cineole (eucalyptol) (8.2%), whilst the EO of C. dioscoridis aerial parts comprises Z-(13,14-epoxy)tetradec-11-en-1-ol acetate (11.6%), γ-elemene (10.2%), tau.-muurolol (7.1%), and cadina-3,9-diene (4.7%). It can be concluded that phytochemical extracts of C. viminalis, E. camaldulensis and C. dioscoridis demonstrated strong to moderate antibacterial effects against the studied plant bacterial pathogens., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Pectobacterium carotovorum subsp. carotovorum strain PccS1, a bacterial pathogen causing soft rot disease of Zantedeschia elliotiana (colored calla), was investigated for virulence genes induced by the host plant. Using a promoter-trap transposon (mariner), we obtained 500 transposon mutants showing kanamycin resistance dependent on extract of Z. elliotiana. One of these mutants, PM86, exhibited attenuated virulence on both Z. elliotiana and Brassica rapa subsp. pekinensis. The growth of PM86 was also reduced in minimal medium (MM), and the reduction was restored by adding plant extract to the MM. The gene containing the insertion site was identified as rplY. The deletion mutant ΔrplY, exhibited reduced virulence, motility and plant cell wall-degrading enzyme production but not biofilm formation. Analysis of gene expression and reporter fusions revealed that the rplY gene in PccS1 is up-regulated at both the transcriptional and the translational levels in the presence of plant extract. Our results suggest that rplY is induced by Z. elliotiana extract and is crucial for virulence in P. carotovorum subsp. carotovorum.

Bacterial pathogens utilize numerous signals to identify the presence of their host and coordinate changes in gene expression that allow for infection. Within plant pathogens, these signals typically include small molecules and/or proteins from their plant hosts and bacterial quorum sensing molecules to ensure sufficient bacterial cell density for successful infection. In addition, bacteria use environmental signals to identify conditions when the host defenses are weakened and potentially to signal entry into an appropriate host/niche for infection. A globin coupled sensor protein (GCS), termed PccGCS, within the soft rot bacterium Pectobacterium carotovorum ssp. carotovorum WPP14 has been identified as an O 2 sensor and demonstrated to alter virulence factor excretion and control motility, with deletion of PccGCS resulting in decreased rotting of a potato host. Using small molecules that modulate bacterial growth and quorum sensing, PccGCS signaling also has been shown to modulate quorum sensing pathways, resulting in the PccGCS deletion strain being more sensitive to plant-derived phenolic acids, which can function as quorum sensing inhibitors, and exhibiting increased N-acylhomoserine lactone (AHL) production. These findings highlight a role for GCS proteins in controlling key O 2 -dependent phenotypes of pathogenic bacteria and suggest that modulating GCS signaling to limit P. carotovorum motility may provide a means to decrease rotting of plant hosts.

Bacterial communities can sense their neighbors, regulating group behaviors in response to cell density and environmental changes. The diversity of signaling networks in a single species has been postulated to allow custom responses to different stimuli; however, little is known about how multiple signals are integrated and the implications of this integration in different ecological contexts. In the plant pathogen Pectobacterium wasabiae (formerly Erwinia carotovora ), two signaling networks-the N-acyl homoserine lactone (AHL) quorum-sensing system and the Gac/Rsm signal transduction pathway-control the expression of secreted plant cell wall-degrading enzymes, its major virulence determinants. We show that the AHL system controls the Gac/Rsm system by affecting the expression of the regulatory RNA RsmB. This regulation is mediated by ExpR2, the quorum-sensing receptor that responds to the P. wasabiae cognate AHL but also to AHLs produced by other bacterial species. As a consequence, this level of regulation allows P. wasabiae to bypass the Gac-dependent regulation of RsmB in the presence of exogenous AHLs or AHL-producing bacteria. We provide in vivo evidence that this pivotal role of RsmB in signal transduction is important for the ability of P. wasabiae to induce virulence in response to other AHL-producing bacteria in multispecies plant lesions. Our results suggest that the signaling architecture in P. wasabiae was coopted to prime the bacteria to eavesdrop on other bacteria and quickly join the efforts of other species, which are already exploiting host resources. IMPORTANCE Quorum-sensing mechanisms enable bacteria to communicate through small signal molecules and coordinate group behaviors. Often, bacteria have various quorum-sensing receptors and integrate information with other signal transduction pathways, presumably allowing them to respond to different ecological contexts. The plant pathogen Pectobacterium wasabiae has two N-acyl homoserine lactone receptors with apparently the same regulatory functions. Our work revealed that the receptor with the broadest signal specificity is also responsible for establishing the link between the main signaling pathways regulating virulence in P. wasabiae This link is essential to provide P. wasabiae with the ability to induce virulence earlier in response to higher densities of other bacterial species. We further present in vivo evidence that this novel regulatory link enables P. wasabiae to join related bacteria in the effort to degrade host tissue in multispecies plant lesions. Our work provides support for the hypothesis that interspecies interactions are among the major factors influencing the network architectures observed in bacterial quorum-sensing pathways., (Copyright © 2017 Valente et al.)

Iron is an important nutrient for the survival and growth of many organisms. In order to survive, iron uptake from the environment must be strictly regulated and maintained to avoid iron toxicity. The ferric uptake regulator protein (Fur) regulates genes involved in iron homeostasis in many bacteria, including phytopathogens. However, to date, the role played by Fur in the biology of Pectobacterium carotovorum subsp. brasiliense (Pcb1692), an important pathogen of potatoes, has not yet been studied. To this end, we used the lambda recombineering method to generate a fur mutant strain of Pcb1692 and assessed the virulence and fitness of the mutant strain. The results showed that production of siderophores in Pcb1692Δfur increased compared to the Pcb1692 wild-type and the complemented strain Pcb1692Δfur-pfur. However, production of N-acyl homoserine lactone (AHLs), biofilm formation, exopolysaccharide (EPS) production, virulence on potato tubers and swimming motility, were all significantly decreased in Pcb1692Δfur compared to the wild-type and complemented Pcb1692Δfur-pfur strains. The Pcb1692Δfur mutant also demonstrated significant sensitivity to oxidative stress when exposed to H2O2. Consistent with phenotypic results, qRT-PCR results demonstrated that Fur down-regulates genes which encode proteins associated with: iron uptake (HasA-extracellular heme-binding protein and Ferrodoxin-AED-0004132), stress response (SodC-superoxide dismutase), plant cell wall degrading enzymes (PrtA and CelV) and motility (FlhC and MotA). We conclude that the ferric uptake regulator protein (Fur) of Pcb1692 regulates traits that are important to host-pathogens interactions.

When exposed to microorganisms, animals use several protective strategies. On one hand, as elegantly exemplified in Drosophila melanogaster, the innate immune system recognizes microbial compounds and triggers an antimicrobial response. On the other hand, behaviors preventing an extensive contact with the microbes and thus reducing the risk of infection have been described. However, these reactions ranging from microbes aversion to intestinal transit increase or food intake decrease have been rarely defined at the molecular level. In this study, we set up an experimental system that allowed us to rapidly identify and quantify food intake decreases in Drosophila larvae exposed to media contaminated with bacteria. Specifically, we report a robust dose-dependent food intake decrease following exposure to the bacteria Erwinia carotovora carotovora strain Ecc15. We demonstrate that this response does not require Imd innate immune pathway, but rather the olfactory neuronal circuitry, the Trpa1 receptor and the evf virulence factor. Finally, we show that Ecc15 induce the same behavior in the invasive pest insect Drosophila suzukii., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Pectobacterium carotovorum ssp. brasiliense 1692 (Pcb1692) is an important emerging pathogen of potatoes causing blackleg in the field and soft rot during post-harvest storage. Blackleg diseases involve the bacterial colonization of vascular tissue and the formation of aggregates, also known as biofilms. To understand the role of quorum sensing in vascular colonization by Pcb1692, we generated a Pcb1692ΔexpI mutant strain. Inactivation of expI led to the reduced production of plant cell wall-degrading enzymes (PCWDEs), the inability to produce acyl homoserine lactone (AHL) and reduced virulence in potato tubers and stems. Complementation of the mutant strain with the wild-type expI gene in trans successfully restored AHL and PCWDE production as well as virulence. Transmission electron microscopy and in vitro motility assays demonstrated hyperpiliation and loss of flagella and swimming motility in the mutant strain compared with the wild-type Pcb1692. Furthermore, we noted that, in the early stages of infection, Pcb1692 wild-type cells had intact flagella which were shed at the later stages of infection. Confocal laser microscopy of PcbΔexpI-inoculated plants showed that the mutant strain tended to aggregate in intercellular spaces, but was unable to transit to xylem tissue. On the contrary, the wild-type strain was often observed forming aggregates within xylem tissue of potato stems. Gene expression analyses confirmed that flagella are part of the quorum sensing regulon, whereas fimbriae and pili appear to be negatively regulated by quorum sensing. The relative expression levels of other important putative virulence genes, such as those encoding different groups of PCWDEs, were down-regulated in the mutant compared with the wild-type strain., (© 2016 BSPP and John Wiley & Sons Ltd.)

Background: Pectobacterium carotovorum subsp. carotovorum (Pcc) is a soft rot bacterium which upon entry into the plant macerates plant tissues by producing plant cell wall degrading enzymes. It has a wide host range which includes carrot, potato, tomato, leafy greens, squash and other cucurbits, onion, green peppers and cassava. During plant-microbe interactions, one of the ways of plant response to pathogen infection is through the small RNA silencing mechanism. Under pathogen attack the plant utilizes microRNAs to regulate gene expression by means of mediating gene silencing at transcriptional and post-transcriptional level. This study aims to assess for the first time, the expression profile of some stress-responsive miRNA and differential expression pattern of their target genes in Arabidopsis thaliana inoculated with Pcc., Materials and Methods: Leaves of five weeks old Arabidopsis thaliana plants were infected with Pcc and the quantitative real time-PCR, was used to investigate after 0, 24, 48 and 72 h post infection, the expression profiling of the stress-responsive miRNAs which include: miR156, miR159, miR169, miR393, miR396 miR398, miR399 and miR408 along with their target genes which include: Squamosa promoter-binding-like protein, myb domain protein 101, nuclear factor Y subunit A8, concanavalin A-like lectin protein kinase, growth regulating factor 4, copper superoxide dismutase, ubiquitin-protein ligase and plantacyanin respectively., Results: The findings showed that the overexpression of 6 miRNAs at 24, 48 and 72 h after infection resulted in the repression of their target genes and the expression of 2 miRNAs didn't affect their target genes., Conclusion: These results provide the first indication of the miRNAs role in response to the infection of Pcc in A. thaliana and open new vistas for a better understanding of miRNA regulation of plant response to Pcc.

Quorum sensing (QS) is a mechanism in which Gram negative bacterial pathogens sense their population density through acyl homoserine lactones (AHLs) and regulate the expression of virulence factors. Enzymatic degradation of AHLs by lactonases, known as quorum quenching (QQ), is thus a potential strategy for attenuating QS regulated bacterial infections. We characterised the QQ activity of soil isolate Lysinibacillus sp. Gs50 and explored its potential for controlling bacterial soft rot of crop plants. Lysinibacillus sp. Gs50 inactivated AHL, which could be restored upon acidification, suggested that inactivation was due to the lactone ring hydrolysis of AHL. Heterologous expression of cloned gene for putative hydrolase (792 bp) designated adeH from Lysinibacillus sp. Gs50 produced a ~29 kDa protein which degraded AHLs of varying chain length. Mass spectrometry analysis of AdeH enzymatic reaction product revealed that AdeH hydrolyses the lactone ring of AHL and hence is an AHL lactonase. Multiple sequence alignment of the amino acid sequence of AdeH showed that it belongs to the metallo- β- lactamase superfamily, has a conserved "HXHXDH" motif typical of AHL lactonases. KM for AdeH for C6HSL was found to be 3.089 μM and the specific activity was 0.8 picomol min-1μg-1. AdeH has not so far been reported from any Lysinibacillus sp. and has less than 40% identity with known AHL lactonases. Finally we found that Lysinibacillus sp. Gs50 can degrade AHL produced by Pectobacterium carotovorum subsp. carotovorum (Pcc), a common cause of soft rot. This QQ activity causes a decrease in production of plant cell wall degrading enzymes of Pcc and attenuates symptoms of soft rot in experimental infection of potato, carrot and cucumber. Our results demonstrate the potential of Lysinibacillus sp. Gs50 as a preventive and curative biocontrol agent., Competing Interests: The authors have declared that no competing interests exist.

Background: Long noncoding RNAs (lncRNAs) represent a class of RNA molecules that are implicated in regulation of gene expression in both mammals and plants. While much progress has been made in determining the biological functions of lncRNAs in mammals, the functional roles of lncRNAs in plants are still poorly understood. Specifically, the roles of long intergenic nocoding RNAs (lincRNAs) in plant defence responses are yet to be fully explored., Results: In this study, we used strand-specific RNA sequencing to identify 1113 lincRNAs in potato (Solanum tuberosum) from stem tissues. The lincRNAs are expressed from all 12 potato chromosomes and generally smaller in size compared to protein-coding genes. Like in other plants, most potato lincRNAs possess single exons. A time-course RNA-seq analysis between a tolerant and a susceptible potato cultivar showed that 559 lincRNAs are responsive to Pectobacterium carotovorum subsp. brasiliense challenge compared to mock-inoculated controls. Moreover, coexpression analysis revealed that 17 of these lincRNAs are highly associated with 12 potato defence-related genes., Conclusions: Together, these results suggest that lincRNAs have potential functional roles in potato defence responses. Furthermore, this work provides the first library of potato lincRNAs and a set of novel lincRNAs implicated in potato defences against P. carotovorum subsp. brasiliense, a member of the soft rot Enterobacteriaceae phytopathogens.

N-acyl-homoserine lactones (AHLs) are metabolites of mostly gram-negative bacteria and are critical signaling molecules in bacterial quorum-sensing systems. At threshold concentrations, AHLs can activate the expression of pathogenic genes and induce diseases. Therefore, reducing AHL concentrations is a key point of disease control in plants. AHL-lactonase, which is expressed by aiiA, is widespread in Bacillus sp and can hydrolyze AHLs. In the present study, we cloned aiiA from Bacillus subtilis by PCR. A plant expression vector of aiiA was constructed and name Pcam-PPP3-aiiA, in which expression of aiiA was controlled by the pathogen-inducible plant promoter PPP3. The recombinant plasmid was transferred into Eucalyptus × urophylla × E. grandis by an Agrobacterium-mediated transformation. PCR and Southern blotting showed that aiiA was successfully integrated into the E. urophylla × E. grandis genome and its expression was induced by Ralstonia solanacearum 12 h after inoculation, as shown by reverse transcription-PCR. The transcription efficacy of aiiA increased 43.88-, 30.65-, and 18.95-fold after inoculation with R. solanacearum, Erwinia carotovora ssp. zeae (Sabet) and Cylindrocladium quinqueseptatum, respectively as shown by RT-real-time PCR. Transgenic E.urophylla × E.grandis expressing the AIIA protein exhibited significantly enhanced disease resistance compared to non-transgenic plants by delaying the onset of wilting and reducing the disease index.

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., (Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Lipopolysaccharides (LPS) are a component of the outer cell surface of almost all Gram-negative bacteria and play an essential role for bacterial growth and survival. Lipopolysaccharides represent typical microbe-associated molecular pattern (MAMP) molecules and have been reported to induce defense-related responses, including the expression of defense genes and the suppression of the hypersensitive response in plants. However, depending on their origin and the challenged plant, LPS were shown to have complex and different roles. In this study we showed that LPS from plant pathogens Pectobacterium atrosepticum and Pectobacterium carotovorum subsp. carotovorum induce common and different responses in A. thaliana cells when compared to those induced by LPS from non-phytopathogens Escherichia coli and Pseudomonas aeruginosa. Among common responses to both types of LPS are the transcription of defense genes and their ability to limit of cell death induced by Pectobacterium carotovorum subsp carotovorum. However, the differential kinetics and amplitude in reactive oxygen species (ROS) generation seemed to regulate defense gene transcription and be determinant to induce programmed cell death in response to LPS from the plant pathogenic Pectobacterium. These data suggest that different signaling pathways could be activated by LPS in A. thaliana cells.

Pectobacterium carotovorum subsp. brasiliense is a newly identified member of the potato soft rot enterobacteriaceae. The pathogenesis of this pathogen is still poorly understood. In this study, an mCherry-P. carotovorum subsp. brasiliense-tagged strain was generated to study P. carotovorum subsp. brasiliense-potato plant interactions. Prior to use, the tagged strain was evaluated for in vitro growth, plasmid stability, and virulence on potato tubers and shown to be similar to the wild type. Four potato cultivars were evaluated for stem-based resistance against P. carotovorum subsp. brasiliense. Confocal laser-scanning microscopy and in vitro viable cell counts showed that P. carotovorum subsp. brasiliense is able to penetrate roots of a susceptible potato cultivar as early as 12 h postinoculation and migrate upward into aerial stem parts. Due to the phenotypic differences observed between tolerant and susceptible cultivars, a comparison of P. carotovorum subsp. brasiliense colonization patterns in these cultivars was undertaken. In the susceptible cultivar, P. carotovorum subsp. brasiliense cells colonized the xylem tissue, forming "biofilm-like" aggregates that led to occlusion of some of the vessels. In contrast, in the tolerant cultivar, P. carotovorum subsp. brasiliense appeared as free-swimming planktonic cells with no specific tissue localization. This suggests that there are resistance mechanisms in the tolerant cultivar that limit aggregation of P. carotovorum subsp. brasiliense in planta and, hence, the lack of symptom development in this cultivar.

Pectobacterium carotovorum subsp. carotovorum is a well-known plant pathogen that causes severe soft rot disease in various crops, resulting in considerable economic loss. To identify pathogenicity-related factors, Chinese cabbage was inoculated with 5314 transposon mutants of P. carotovorum subsp. carotovorum Pcc21 derived using Tn5 transposon mutagenesis. A total of 35 reduced-virulence or avirulent mutants were isolated, and 14 loci were identified. The 14 loci could be functionally grouped into nutrient utilization (pyrD, purH, purD, leuA and serB), production of plant cell-wall-degrading enzymes (PCWDEs) (expI, expR and PCC21&#95;023220), motility (flgA, fliA and flhB), biofilm formation (expI, expR and qseC), susceptibility to antibacterial plant chemicals (tolC) and unknown function (ECA2640). Among the 14 genes identified, qseC, tolC and PCC21&#95;023220 are novel pathogenicity factors of P. carotovorum subsp. carotovorum involved in biofilm formation, phytochemical resistance and PCWDE production, respectively.

Pectobacterium species are enterobacterial plant-pathogens that cause soft rot disease in diverse plant species. Unlike hemi-biotrophic plant pathogenic bacteria, the type III secretion system (T3SS) of Pectobacterium carotovorum subsp. carotovorum (P. carotovorum) appears to secrete only one effector protein, DspE. Previously, we found that the T3SS regulator HrpL and the effector DspE are required for P. carotovorum pathogenesis on leaves. Here, we identified genes up-regulated by HrpL, visualized expression of dspE in leaves, and established that DspE causes host cell death. DspE required its full length and WxxxE-like motifs, which are characteristic of the AvrE-family effectors, for host cell death. We also examined expression in plant leaves and showed that hrpL is required for the expression of dspE and hrpN, and that the loss of a functional T3SS had unexpected effects on expression of other genes during leaf infection. These data support a model where P. carotovorum uses the T3SS early in leaf infection to initiate pathogenesis through elicitation of DspE-mediated host cell death.

Innate immunity plays an important role in combating microbial infection in animals. During bacterial infection in Drosophila melanogaster gut, Dual oxidase (Duox) generates reactive oxygen species (ROS) to fight against the infected microbes. Concurrently, antioxidant systems eliminate residual ROS and protect the hosts. Here we found that Drosophila melanogaster Peroxiredoxin V (dPrxV) is an immune-related antioxidant enzyme which maintains intestinal redox homeostasis. dPrxV was highly expressed in gut and induced by the oral infection of Erwinia carotovora carotovora. dPrxV expression was increased by the gut-specific Duox overexpression but decreased by Duox inhibition. Moreover, dPrxV expression was mediated by the JNK/FOXO signaling and dPrxV mutant reduced survival after gut infection. These results suggest that JNK/FOXO mediated dPrxV expression plays a critical role in Drosophila melanogaster gut during bacterial infection in protecting the host gut epithelial cells from oxidative damage., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Pectobacterium carotovorum subsp. carotovorum causes soft rot disease in various plants, including Chinese cabbage. The simple extracellular leucine-rich repeat (eLRR) domain proteins have been implicated in disease resistance. Rice leucine-rich repeat protein (OsLRP), a rice simple eLRR domain protein, is induced by pathogens, phytohormones, and salt. To see whether OsLRP enhances disease resistance to bacterial soft rot, OsLRP was introduced into Chinese cabbage by Agrobacterium-mediated transformation. Two independent transgenic lines over-expressing OsLRP were generated and further analyzed. Transgenic lines over-expressing OsLRP showed enhanced disease resistance to bacterial soft rot compared to non-transgenic control. Bacterial growth was retarded in transgenic lines over-expressing OsLRP compared to non-transgenic controls. We propose that OsLRP confers enhanced resistance to bacterial soft rot. Monitoring expression of defense-associated genes in transgenic lines over-expressing OsLRP, two different glucanases and Brassica rapa polygalacturonase inhibiting protein 2, PDF1 were constitutively activated in transgenic lines compared to non-transgenic control. Taken together, heterologous expression of OsLRP results in the activation of defense response and enhanced resistance to bacterial soft rot.

Objective: To investigate functions of flgKpcc gene in Pectobacterium carotovorum subsp. carotovorum (P. c. c)., Methods: The gene knock-out mutant deltaflgKpcc and complemented strain deltaflgKpcc-KH were generated by biparental mating and their phenotypes including cell morphology, motility, pathogenic factors, and pathogenicity were investigated., Results: Non-flagellum, cell precipitation in the culture and significantly attenuated motility on 0.3% semisolid medium were observed in deltaflgKpcc compared to Pectobacterium carotovorum subsp. carotovorum S1. In addition, significant decrease in cellulase and protease activity, biofilm formation, and pathogenicity on host plant were found in deltaflgKpcc. While there were no apparent difference in growth rate in vitro, deltaflgKpcc-KH, the complementation strain, restored the phenotype of deltaflgKpcc to the wild type level., Conclusion: The gen of flgKpcc not only influences the cell motility, but also pathogenic factors to lead to the decreased pathogenicity in Pectobacterium carotovorum subsp. Carotovorum.

Bacterial diseases of weeds horsetail common (Equisetum arvense L.) were revealed in the crops of wheat and soya in the fields of Kyiv and Vinnitsia Regions of Ukraine. The distinctive symptoms of bacterial affections on the root neck, on stalks of vegetative and spore shoots, on twigs were brown, dark brown or almost black necrotic spots of oblong form. The necroses increased in size, embraced the stalks. The stalks broke, the plants dried up. Patterns of affected plants, isolated and identified phytopathogenic bacteria Pseudomonas syringae, Pectobacterium carotovorum subsp. carotovorum, Pantoea agglomerans and Curtobacterium sp. were analyzed These bacteria caused pathological process on the horsetail common, wheat and soy under the conditions of artificial inoculation. The composition of bacteria species was different in different years depending on temperature conditions of vegetative period.

Two biotic stress resistance related genes from the full-length cDNA library of Brassica rapa cv. Osome were identified from EST analysis and determined to be pathogenesis-related (PR) 12 Brassica defensin-like family protein (BrDLFP) and PR-10 Brassica Betv1 allergen family protein (BrBetv1AFP) after sequence analysis and homology study with other stress resistance related same family genes. In the expression analysis, both genes expressed in different organs and during all developmental growth stages in healthy plants. Expression of BrDLFP significantly increased and BrBetv1AFP gradually decreased after infection with Pectobacterium carotovorum subsp. carotovorum in Chinese cabbage. Expression of these two genes significantly changed after cold, salt, drought and ABA stress treatments. These two PR genes may therefore be involved in the plant resistance against biotic and abiotic stresses.

Plant cells can be sensitized toward a subsequent pathogen attack by avirulent pathogens or by chemicals such as β-aminobutyric acid (BABA). This process is called priming. Using a reverse genetic approach in Arabidopsis thaliana, we demonstrate that the BABA-responsive L-type lectin receptor kinase-VI.2 (LecRK-VI.2) contributes to disease resistance against the hemibiotrophic Pseudomonas syringae and the necrotrophic Pectobacterium carotovorum bacteria. Accordingly, LecRK-VI.2 mRNA levels increased after bacterial inoculation or treatments with microbe-associated molecular patterns (MAMPs). We also show that LecRK-VI.2 is required for full activation of pattern-triggered immunity (PTI); notably, lecrk-VI.2-1 mutants show reduced upregulation of PTI marker genes, impaired callose deposition, and defective stomatal closure. Overexpression studies combined with genome-wide microarray analyses indicate that LecRK-VI.2 positively regulates the PTI response. LecRK-VI.2 is demonstrated to act upstream of mitogen-activated protein kinase signaling, but independently of reactive oxygen production and Botrytis-induced kinase1 phosphorylation. In addition, complex formation between the MAMP receptor flagellin sensing2 and its signaling partner brassinosteroid insensitive1-associated kinase1 is observed in flg22-treated lecrk-VI.2-1 mutants. LecRK-VI.2 is also required for full BABA-induced resistance and priming of PTI. Our work identifies LecRK-VI.2 as a novel mediator of the Arabidopsis PTI response and provides insight into molecular mechanisms governing priming.

Solanum tuberosum plants were transformed with three genetic constructions expressing the Nicotiana tabacum AP24 osmotine, Phyllomedusa sauvagii dermaseptin and Gallus gallus lysozyme, and with a double-transgene construction expressing the AP24 and lysozyme sequences. Re-transformation of dermaseptin-transformed plants with the AP24/lysozyme construction allowed selection of plants simultaneously expressing the three transgenes. Potato lines expressing individual transgenes or double- and triple-transgene combinations were assayed for resistance to Erwinia carotovora using whole-plant and tuber infection assays. Resistance levels for both infection tests compared consistently for most potato lines and allowed selection of highly resistant phenotypes. Higher resistance levels were found in lines carrying the dermaseptin and lysozyme sequences, indicating that theses proteins are the major contributors to antibacterial activity. Similar results were obtained in tuber infection tests conducted with Streptomyces scabies. Plant lines showing the higher resistance to bacterial infections were challenged with Phytophthora infestans, Rhizoctonia solani and Fusarium solani. Considerable levels of resistance to each of these pathogens were evidenced employing semi-quantitative tests based in detached-leaf inoculation, fungal growth inhibition and in vitro plant inoculation. On the basis of these results, we propose that stacking of these transgenes is a promising approach to achieve resistance to both bacterial and fungal pathogens., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Pectobacterium carotovorum and Pectobacterium atrosepticum are dreadful causal agents of potato soft rot. Actually, there are no efficient bactericides used to protect potato against Pectobacterium spp. Biological control using actinobacteria could be an interesting approach to manage this disease. Thus, two hundred actinobacteria isolated from Moroccan habitats were tested for their ability to inhibit in vitro 4 environmental Pectobacterium strains and the two reference strains (P. carotovorum CFBP 5890 and P. atrosepticum CFBP 5889). Eight percent of these isolates were active against at least one of the tested pathogens and only 2% exhibited an antimicrobial activity against all tested Pectobacterium strains. Four bioactive isolates having the greatest pathogen inhibitory capabilities and classified as belonging to the genus Streptomyces species through 16S rDNA analysis were subsequently tested for their ability to reduce in vivo soft rot symptoms on potato slices of Bintje, Yukon Gold, Russet and Norland cultivars caused by the two pathogens P. carotovorum and P. atrosepticum. This test was carried out by using biomass inoculums and culture filtrate of the isolates as treatment. Among these, strain Streptomyces sp. OE7, reduced by 65-94% symptom severity caused by the two pathogens on potato slices. Streptomyces OE7 showed a potential for controlling soft rot on potato slices and could be useful in an integrated control program against potato soft rot pathogens in the objective to reduce treatments with chemical compounds.

Pectobacterium carotovorum (formerly Erwinia carotovora ssp. carotovora) is a phytopathogenic bacterium that causes soft rot disease, characterized by water-soaked soft decay, resulting from the action of cell wall-degrading exoenzymes secreted by the pathogen. Virulence in soft rot bacteria is regulated by environmental factors, host and bacterial chemical signals, and a network of global and gene-specific bacterial regulators. We isolated a mini-Tn5 mutant of P. carotovorum that is reduced in the production of extracellular pectate lyase, protease, polygalacturonase and cellulase. The mutant is also decreased in virulence as it macerates less host tissues than its parent and is severely impaired in multiplication in planta. The inactivated gene responsible for the reduced virulent phenotype was identified as corA. CorA, a magnesium/nickel/cobalt membrane transporter, is the primary magnesium transporter for many bacteria. Compared with the parent, the CorA(-) mutant is cobalt resistant. The mutant phenotype was confirmed in parental strain P. carotovorum by marker exchange inactivation of corA. A functional corA(+) DNA from P. carotovorum restored exoenzyme production and pathogenicity to the mutants. The P. carotovorum corA(+) clone also restored motility and cobalt sensitivity to a CorA(-) mutant of Salmonella enterica. These data indicate that CorA is required for exoenzyme production and virulence in P. carotovorum., (© 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.)

It is shown for the first time that population heterogeneity of Pectobacterium carotovorum subsp. carotovorum is applicable to a wide range of strains and therefore is a universal characteristic. Using the method of specific selection with the help of carotovoricins which are identical to the phage tails a set of population dissociants of different types was obtained, due to the fact that S-LPS is the part of the cell wall which contains their attachment sites. It was determined that changes in S-lipopolysaccharides lead to the formation of SR-, R-forms of P. carotovorum. A suggestion is made that the changes in the surface structures of dissociants have a significant impact on secretion types II and III--the main pathogenicity factor of some bacteria. The results presented are a prerequisite for studying the direction, the reasons for dissociation process and its impact on the pathogenicity of P. carotovorum.

Quorum sensing (QS) is a population density-dependent regulatory mechanism in which gene expression is coupled to the accumulation of a chemical signaling molecule. QS systems are widespread among the plant soft-rotting bacteria. In Pectobacterium carotovorum, at least two QS systems exist being specified by the nature of chemical signals involved. QS in Pectobacterium carotovorum uses N-acylhomoserine lactone (AHL) based, as well as autoinducer-2 (AI-2) dependent signaling systems. This review will address the importance of the QS in production of virulence factors and interaction of QS with other regulatory systems in Pectobacterium carotovorum.