Jean M. Bonasera

Downstream Divergence of the Ethylene Signaling Pathway for Harpin-Stimulated Arabidopsis Growth and Insect Defense

Ethylene (ET) signal transduction may regulate plant growth and defense, depending on which components are recruited into the pathway in response to different stimuli. We report here that the ET pathway controls both insect resistance (IR) and plant growth enhancement (PGE) in Arabidopsis (Arabidopsis thaliana) plants responding to harpin, a protein produced by a plant pathogenic bacterium. PGE may result from spraying plant tops with harpin or by soaking seeds in harpin solution; the latter especially enhances root growth. Plants treated similarly develop resistance to the green peach aphid (Myzus persicae). The salicylic acid pathway, although activated by harpin, does not lead to PGE and IR. By contrast, PGE and IR are induced in both wild-type plants and genotypes that have defects in salicylic acid signaling. In response to harpin, levels of jasmonic acid (JA) decrease, and the COI1 gene, which is indispensable for JA signal transduction, is not expressed in wild-type plants. However, PGE and IR are stimulated in the JA-resistant mutant jar1-1. In the wild type, PGE and IR develop coincidently with increases in ET levels and the expression of several genes essential for ET signaling. The ET receptor gene ETR1 is required because both phenotypes are arrested in the etr1-1 mutant. Consistently, inhibition of ET perception nullifies the induction of both PGE and IR. The signal transducer EIN2 is required for IR, and EIN5 is required for PGE because IR and PGE are impaired correspondingly in the ein2-1 and ein5-1 mutants. Therefore, harpin activates ET signaling while conscribing EIN2 and EIN5 to confer IR and PGE, respectively.

Harpin-elicited hypersensitive cell death and pathogen resistance require the NDR1 and EDS1 genes

Abstract Plants sprayed with harpin, a bacterial protein that induces hypersensitive cell death (HCD), develop systemic acquired resistance (SAR) without macroscopic necrosis. HCD sometimes accompanies the development of resistance conferred by resistance ( R ) genes. In Arabidopsis , some R genes require one or both of the signalling components NDR1 and EDS1 for function. This study addresses whether HCD, NDR1 and EDS1 are required for induction of SAR by harpin. When Arabidopsis and tobacco leaves were sprayed with harpin, microscopic hypersensitive response (micro-HR) lesions developed. Systemic expression of PR genes and the development of resistance were accompanied by micro-HR, except in the ndr 1-1 mutant, in which harpin induced micro-HR without the development of resistance or expression of the PR -1 gene. Cell death and resistance did not occur following treatment with harpin in plants that could not accumulate salicylic acid. Harpin also failed to induce resistance in Arabidopsis eds 1-1 mutants. Therefore, harpin-induced resistance seems to develop concomitantly with cell death and resistance requires NDR 1 and EDS 1.

PR genes of apple: identification and expression in response to elicitors and inoculation with Erwinia amylovora

BackgroundIn the past decade, much work has been done to dissect the molecular basis of the defence signalling pathway in plants known as Systemic Acquired Resistance (SAR). Most of the work has been carried out in model species such as Arabidopsis, with little attention paid to woody plants. However within the range of species examined, components of the pathway seem to be highly conserved. In this study, we attempted to identify downstream components of the SAR pathway in apple to serve as markers for its activation.ResultsWe identified three pathogenesis related (PR) genes from apple, PR-2, PR-5 and PR-8, which are induced in response to inoculation with the apple pathogen, Erwinia amylovora, but they are not induced in young apple shoots by treatment with known elicitors of SAR in herbaceous plants. We also identified three PR-1-like genes from apple, PR-1a, PR-1b and PR-1c, based solely on sequence similarity to known PR-1 genes of model (intensively researched) herbaceous plants. The PR-1-like genes were not induced in response to inoculation with E. amylovora or by treatment with elicitors; however, each showed a distinct pattern of expression.ConclusionFour PR genes from apple were partially characterized. PR-1a, PR-2, PR-5 and PR-8 from apple are not markers for SAR in young apple shoots. Two additional PR-1-like genes were identified through in-silico analysis of apple ESTs deposited in GenBank. PR-1a, PR-1b and PR-1c are not involved in defence response or SAR in young apple shoots; this conclusion differs from that reported previously for young apple seedlings.

Apple Proteins that Interact with DspA/E, a Pathogenicity Effector of Erwinia amylovora, the Fire Blight Pathogen

The disease-specific (dsp) gene dspA/E of Erwinia amylovora encodes an essential pathogenicity effector of 198 kDa, which is critical to the development of the devastating plant disease fire blight. A yeast two-hybrid assay and in vitro protein pull-down assay demonstrated that DspA/E interacts physically and specifically with four similar putative leucine-rich repeat (LRR) receptor-like serine/threonine kinases (RLK) from apple, an important host of E. amylovora. The genes encoding these four DspA/E-interacting proteins of Malus xdomestica (DIPM1 to 4) are conserved in all genera of hosts of E. amylovora tested. They also are conserved in all cultivars of apple tested that range in susceptibility to fire blight from highly susceptible to highly resistant. The four DIPMs have been characterized, and they are expressed constitutively in host plants. In silico analysis indicated that the DIPMs have similar sequence structure and resemble LRR RLKs from other organisms. Evidence is presented for direct physical interaction between DspA/E and the apple proteins encoded by the four identified clones, which may act as susceptibility factors and be essential to disease development. Knowledge of DIPMs and the interaction with DspA/E thus may facilitate understanding of fire blight development and lead to new approaches to control of disease.

OEM--a new medium for rapid isolation of onion-pathogenic and onion-associated bacteria.

Onions (Allium cepa L.) are plagued by a number of bacterial pathogens including Pantoea ananatis, P. agglomerans, Burkholderia cepacia, Enterobacter cloacae, Pectobacterium carotovorum subsp. carotovorum, Xanthomonas axonopodis pv. axonopodis and several Pseudomonas spp. We developed a semi-selective medium, termed onion extract medium (OEM), to selectively and rapidly isolate bacteria pathogenic to and associated with onions and onion-related samples including bulbs, seeds, sets, transplant seedlings, soil and water. Most strains of interest grow sufficiently on OEM in 24h at 28°C for tentative identification based on colony morphology, facilitating further characterization by microbiological and/or molecular means.

Infection of Onion Leaves by Pantoea ananatis Leads to Bulb Infection

Pantoea ananatis has been identified as a cause of center rot of onion. In the field, onion leaves can become infected with P. ananatis and lead to leaf blight. Infected bulbs often are detected only after harvest; however, it has not been demonstrated experimentally that leaf infection by P. ananatis can lead to bulb infection. In this study, onion leaf infection by P. ananatis leading to bulb infection was investigated. Of 18 strains of P. ananatis isolated from symptomatic onion bulbs grown in New York, 14 were pathogenic in bulb and leaf tissue. Pathogenic strains of P. ananatis caused nonmacerated, yellow-brown coloration in fleshy bulb scales following inoculation of bulbs and incubation for 2 days at 28°C. Subepidermal inoculation of onion leaves with pathogenic strains of P. ananatis resulted in gray-white foliar lesions that extended acropetally and basipetally from the points of inoculation. In all, 16% of leaf lesions extended to the onion neck and 11% continued into the bulbs, which developed nonmacerated, yellow-brown scales. Bacteria recovered from the leading edges of lesions had microbiological and molecular characteristics of P. ananatis. This is the first experimental evidence that infection of onion leaves by P. ananatis can lead to bulb infection.

First report of enterobacter bulb decay of onions caused by Enterobacter cloacae in New York.

During the summer of 2010, onions (Allium cepa L.) of several cultivars growing in muck-land soils in Orange, Genesee, Orleans, and Oswego counties of New York exhibited leaf dieback and bulb decay consistent with disease symptoms caused by Enterobacter cloacae as described previously (1,3,4). Isolations of bacteria from symptomatic tissues and muck soil were made using onion extract medium (OEM), which contains extracts of autoclaved onions, salts, and inhibitors of fungi and gram-positive bacteria. Some presumptive strains of E. cloacae were isolated; 5 from symptomatic onions growing in Genesee County, 2 from muck-land soil, and 27 from bulbs stored for ~2.5 months in a farm storage facility in Oswego County. Tentative identification was based on colony morphology (convex, cream-color colonies, 2 to 3 mm in diameter following incubation at 28°C for 1 day on OEM), which was similar to the morphology of reference strains of E. cloacae ATCC 23355, ATCC 13047, and strain 310 (gift of H. F. Schwartz, which was derived from reference 4; personal communication). Strains were gram-negative rods, negative for oxidase and indole, positive for nitrate reductase and catalase; produced acid from glucose aerobically and anaerobically. Also, all strains produced PCR products from the 16S-23S internal transcribed spacer (ITS) DNA region of the predicted sizes using primers T5A and T3B designed for identification of E. cloacae (2). The growth of eight of the isolated strains and strains ATTC 23355 and 310 were evaluated on several carbon sources with RapiD 20E test strips (bio Mérieux, Inc, Durham, NC). All strains were positive for β-d-galactosidase, ornithine decarboxylase, utilization of citrate and malonate, and production of acetoin. Hydrolysis of esculin by β-glucosidase differed among the eight. All strains were negative for lysine decarboxylase, urease, para-phenylalanine deaminase, indole, and oxidase. All produced acid from arabinose, xylose, rhamnose, cellobiose, melibiose, saccharose, trehalose, raffinose, and glucose; no strains produced acid from adonitol. These characteristics are consistent with published data for E. cloacae. Surface-disinfested onion bulbs and sets were inoculated with 50 to 100 μl of bacterial suspensions containing ~108 CFU/ml, injected with hypodermic needles and syringes, and incubated at 37°C for 2 weeks. Bisected onions revealed dry brown discoloration in each of the four bulbs and sets that had been inoculated with each presumptive strain. Symptoms were indistinguishable from those apparent in onions inoculated with the authentic strains mentioned. Strains recovered on OEM were identified as E. cloacae based on the stated biochemical properties and analysis of the 16S rRNA gene amplified by PCR as above. The sequence of the amplicon from the isolated strains was identical to that of reference strains ATCC 23355 and 310. Amplicon sequences of the 16S rRNA gene of New York strains Ecl3, Ecl6, and Ecl7 were deposited in GenBank as JF832951, JF832952, and JF832953, respectively. The strains were accessioned as ATCC BAA-2271, ATCC BAA-2272, and ATCC BAA-2273, respectively. To our knowledge, this is the first published report of E. cloacae causing Enterobacter bulb decay of onion in New York. References: (1) A. L. Bishop and R. M. Davis. Plant Dis. 74:692, 1990. (2) M. M. Clementino et al. J. Clin. Microbiol. 39:3865, 2004. (3) B. K. Schroeder and L. J. du Toit. Plant Dis. 93:323, 2009. (4) H. F. Schwartz and K. Otto. Plant Dis. 84:808, 2000.

Identification of bacteria pathogenic to or associated with onion (Allium cepa) based on sequence differences in a portion of the conserved gyrase B gene.

We have developed a method for the identification of Gram-negative bacteria, particularly members of the Enterobacteriaceae, based on sequence variation in a portion of the gyrB gene. Thus, we identified, in most cases to species level, over 1000 isolates from onion bulbs and leaves and soil in which onions were grown.

Lactic Acid Bacteria Cause a Leaf Blight and Bulb Decay of Onion (Allium cepa)

Several members of the lactic acid bacteria group were isolated from diseased onion plants and bulbs. Based on growth characteristics and sequence analysis of 16S rRNA and rpoA genes, the strains were identified as Lactococcus lactis, Lactobacillus plantarum, and three species of Leuconostoc, i.e., citreum, mesenteroides, and pseudomesenteroides. Pathogenic potential to onion leaves and mature onion bulbs was assessed. L. plantarum and all three Leuconostoc species caused symptoms in both leaves and bulbs. L. lactis caused scale discoloration in bulbs but failed to cause lesions on leaves. Leuconostoc citreum caused bulb decay in 7 days at 18°C as well as 37°C. This is the first report of a group of gram-positive bacteria able to cause disease in onion leaves.

PCR Primers for Detection of Pantoea ananatis, Burkholderia spp., and Enterobacter sp. from Onion

Bacterial decays of onion bulbs cause sporadic and sometimes serious losses to onion (Allium cepa). In New York, three groups of bacteria were identified as problematic: Burkholderia spp., Pantoea ananatis, and Enterobacter spp. To aid in efficient detection and diagnosis of these pathogens, pairs of specific polymerase chain reaction primers were designed and validated, based on a strategy that utilized various genome sequences now available in public databases. Primer pairs were tested against numerous strains of target bacteria, closely related bacteria, and other onion-pathogenic bacteria. Each primer pair yielded a single, apparently highly specific amplicon from aqueous suspensions of the target bacteria. Minimum sensitivities were approximately 103 CFU per 25-μl reaction mixture for all three primer pairs.