Copper Resistance Gene Homologs in Pathogenic and Saprophytic Bacterial Species from Tomato

Copper-resistant strains of Xanthomonas campestris pv. vesicatoria, Pseudomonas cichorii, Pseudomonas putida, Pseudomonas fluorescens, and a yellow Pseudomonas sp. were isolated from tomato plants or seeds. In Southern hybridizations, DNA from each strain showed homology with the copper resistance (cop) operon previously cloned from Pseudomonas syringae pv. tomato PT23. Homology was associated with plasmid and chromosomal DNA in X. compestris pv. vesicatoria, P. putida, and the yellow Pseudomonas sp. Homology was detected only in the chromosomal DNA of P. cichorii and P. fluorescens. Homology with cop was also detected in chromosomal DNA from copper-sensitive strains of P. cichorii, P. fluorescens, and P. syringae pv. tomato, suggesting that the cop homolog may be indigenous to certain Pseudomonas species and have some function other than copper resistance. No homology was detected in DNA from a copper-sensitive X. campestris pv. vesicatoria strain. Copper-inducible protein products were detected in each copper-resistant bacterium by immunoblot analysis with antibodies raised to the CopB protein from the cop operon. The role of the homologous DNA in copper resistance was confirmed for the X. campestris pv. vesicatoria strain by cloning and transferring the cop homolog to a copper-sensitive strain of X. campestris pv. vesicatoria. The possibility and implications of copper resistance gene exchange between different species and genera of pathogenic and saprophytic bacteria on tomato plants are discussed.     

Reservoir Weed Hosts of Tomato Yellow Leaf Curl Begomovirus from Tanzania

A survey was initiated to detect tomato yellow leaf curl virus (TYLCV) and identify its reservoir weed hosts in six regions (Arusha, Morogoro, Dodoma, Iringa, Kilimanjaro and Dar es Salaam) in Tanzania. Three farms were randomly selected in each region. Assessment of TYLCV incidence was done by relating the number of infected tomato plants to the total number of plants assessed along a diagonal in five quadrants measuring 4m ‐ 4m in size (one at each corner of the farm and one at the centre). Disease severity was scored on a scale of 0 to 4 (where 0 = no symptoms and 4 = very severe symptoms). Within and outside each farm, weeds showing TYLCV-like symptoms were collected and either squash-blotted, dot-blotted or both on nylon membranes. The membranes were hybridized with DIG-labelled probe synthesized for the detection of TYLCV from Sardinia (TYLCV-Sar) following standard protocols. Selected plant species were experimentally inoculated with screenhouse cultures of TYLCV representative isolates from the six regions using Bemisia tabaci to determine their host status. Results indicated that TYLCV incidence and severity were significantly higher (P = 0.05) in Dodoma region than the rest of the regions. In Iringa region, the incidence and severity of TYLCV were the lowest of all regions. TYLCV was detected in 12 of the 17 dot-blotted samples and in all the 21 squashed samples using the non-radioactively labelled riboprobes. Similarly, five plant species (Capsicum annuum, Datura stramonium, Lycopersicon esculentum, Nicotiana glutionsa and N. tabacum) tested in the screenhouse were infected by the six TYLCV isolates used. It is recommended that weeds within and outside tomato farms be removed to eliminate or reduce sources of virus inoculum. The dot and squash blot techniques are convenient for field detection of the virus, and are especially useful for the detection of early and latent infections so that management strategies can be initiated and implemented.     

Suppression by ABA of salicylic acid and lignin accumulation and the expression of multiple genes,in <Emphasis Type="Italic">Arabidopsis</Emphasis> infected with <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tomato</Emphasis>

Abscisic acid (ABA) has been implicated in determining the outcome of interactions between many plants and their pathogens. We had previously shown that increased concentrations of ABA within leaves of Arabidopsis induced susceptibility towards an avirulent strain of Pseudomonas syringae pathovar (pv.) tomato. We now show that ABA induces susceptibility via suppression of the accumulation of components crucial for a resistance response. Lignin and salicylic acid concentrations in leaves were increased during a resistant interaction but reduced when plants were treated with ABA. The reduction in lignin and salicylic acid production was independent of the development of the hypersensitive response (HR), indicating that, in this host-pathogen system, HR is not required for resistance. Genome-wide gene expression analysis using microarrays showed that treatment with ABA suppressed the expression of many defence-related genes, including those important for phenylpropanoid biosynthesis and those encoding resistance-related proteins. Together, these results show that resistance induction in Arabidopsis to an avirulent strain of P. syringae pv. tomato is regulated by ABA. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Ribosomal protein QM/RPL10 positively regulates defence and protein translation mechanisms during nonhost disease resistance

Ribosomes play an integral part in plant growth, development, and defence responses. We report here the role of ribosomal protein large (RPL) subunit QM/RPL10 in nonhost disease resistance. The RPL10-silenced Nicotiana benthamiana plants showed compromised disease resistance against nonhost pathogen Pseudomonas syringae pv. tomato T1. The RNA-sequencing analysis revealed that many genes involved in defence and protein translation mechanisms were differentially affected due to silencing of NbRPL10. Arabidopsis AtRPL10 RNAi and rpl10 mutant lines showed compromised nonhost disease resistance to P. syringae pv. tomato T1 and P. syringae pv. tabaci. Overexpression of AtRPL10A in Arabidopsis resulted in reduced susceptibility against host pathogen P. syringae pv. tomato DC3000. RPL10 interacts with the RNA recognition motif protein and ribosomal proteins RPL30, RPL23, and RPS30 in the yeast two-hybrid assay. Silencing or mutants of genes encoding these RPL10-interacting proteins in N. benthamiana or Arabidopsis, respectively, also showed compromised disease resistance to nonhost pathogens. These results suggest that QM/RPL10 positively regulates the defence and translation-associated genes during nonhost pathogen infection.     

New disease resistance genes in soybean against Pseudomonas syringae pv glycinea: evidence that one of them interacts with a bacterial elicitor

Summary Soybean [Glycine max (L.) Merr.] cultivars Flambeau and Merit differed in their resistance to Pseudomonas syringae pv glycinea (Psg) race 4, carrying each of four different avirulence (avr) genes cloned from Psg or the related bacterium, Pseudomonas syringae pv tomato. Segregation data for F₂ and F₃ progeny of Flambeau x Merit crosses indicated that single dominant and nonallelic genes account for resistance to Psg race 4, carrying avirulence genes avrA, avrB, avrC, or avrD. Segregants were also recovered that carried all four or none of the disease resistance genes. One of the disease resistance genes (Rpg1, complementing bacterial avirulence gene B) had been described previously, but the other three genes — designated Rpg2, Rpg3, and Rpg4 — had not here to fore been defined. Rpg3 and Rpg4 are linked (40.5 ± 3.2 recombination units). Rpg4 complements avrD, cloned from Pseudomonas syringae pv tomato, but a functional copy of this avirulence gene has not thus far been observed in Pseudomonas syringae pv glycinea. Resistance gene Rpg4 therefore may account in part for the resistance of soybean to Pseudomonas syringae pv tomato and other pathogens harboring avrD.     

Pto3 and Pto4: novel genes from Lycopersicon hirsutum var. glabratum that confer resistance to Pseudomonas syringae pv tomato

Accessions of wild Lycopersicon germplasm were screened for resistance to Pseudomonas syringae pv tomato (P.s. tomato). Resistance to both race-0 and race-1 strains of P.s. tomato was identified in L. pimpinellifolium, L. peruvianum and L. hirsutum var. glabratum. Resistance to race-0 derived from L. hirsutum var. glabratum (Pto3) appeared to be inherited independently of Pto1 and Pto2. Filial and backcross generations derived from interspecific crosses between L. esculentum and L. hirsutum var. glabratum revealed that Pto3 resistance was inherited in a complex fashion and was incompletely dominant under conditions of high bacteria inocula. Resistance to P.s. tomato race-1 (Pto4) was also identified in L. hirsutum var. glabratum. Pto3 and Pto4 segregated independently of each other.     

Status of Streptomycin Resistance Development in Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola in China and their Resistance Characters

Rice leaves with bacterial blight or bacterial leaf streak symptoms were collected in southern China in 2007 and 2008. Five hundred and thirty‐four single‐colony isolates of Xanthomonas oryzae pv. oryzae and 827 single‐colony isolates of Xanthomonas oryzae pv. oryzicola were obtained and tested on plates for sensitivity to streptomycin. Four strains (0.75%) of X. oryzae pv. oryzae isolated from the same county of Province Yunnan were resistant to streptomycin, and the resistance factor (the ratio of the mean median effective concentration inhibiting growth of resistant isolates to that of sensitive isolates) was approximately 226. The resistant isolate also showed streptomycin resistance in vivo. In addition to resistant isolates, isolates of less sensitivity were also present in the population of X. oryzae pv. oryzae from Province Yunnan. However, no isolates with decreased streptomycin‐sensitivity were obtained from the population of X. oryzae pv. oryzicola. Mutations in the rpsL (encoding S12 protein) and rrs genes (encoding 16S rRNA) and the presence of the strA gene accounting for streptomycin resistance in other phytopathogens or animal and human pathogenic bacteria were examined on sensitive and resistant strains of X. oryzae pv. oryzae by polymerase chain reaction amplification and sequencing. Neither the presence of the strA gene nor mutations in the rpsL or rrs were found, suggesting that different resistance mechanisms are involved in the resistant isolates of X. oryzae pv. oryzae.     

Natural Variation for Responsiveness to flg22, flgII-28, and csp22 and Pseudomonas syringae pv. tomato in Heirloom Tomatoes

Tomato (Solanum lycopersicum L.) is susceptible to many diseases including bacterial speck caused by Pseudomonas syringae pv. tomato. Bacterial speck disease is a serious problem worldwide in tomato production areas where moist conditions and cool temperatures occur. To enhance breeding of speck resistant fresh-market tomato cultivars we identified a race 0 field isolate, NC-C3, of P. s. pv. tomato in North Carolina and used it to screen a collection of heirloom tomato lines for speck resistance in the field. We observed statistically significant variation among the heirloom tomatoes for their response to P. s. pv. tomato NC-C3 with two lines showing resistance approaching a cultivar that expresses the Pto resistance gene, although none of the heirloom lines have Pto. Using an assay that measures microbe-associated molecular pattern (MAMP)-induced production of reactive oxygen species (ROS), we investigated whether the heirloom lines showed differential responsiveness to three bacterial-derived peptide MAMPs: flg22 and flgII-28 (from flagellin) and csp22 (from cold shock protein). Significant differences were observed for MAMP responsiveness among the lines, although these differences did not correlate strongly with resistance or susceptibility to bacterial speck disease. The identification of natural variation for MAMP responsiveness opens up the possibility of using a genetic approach to identify the underlying loci and to facilitate breeding of cultivars with enhanced disease resistance. Towards this goal, we discovered that responsiveness to csp22 segregates as a single locus in an F₂ population of tomato.     

Indigenous plasmids in Pseudomonas syringae pv. tomato: conjugative transfer and role in copper resistance

Twenty strains of Pseudomonas syringae pv. tomato were examined for the presence of plasmid DNA. P. syringae pv. tomato plasmids were grouped into five size classes: class A ranged from 95 to 103 kilobases (kb); class B ranged from 71 to 83 kb; class C ranged from 59 to 67 kb; class D ranged from 37 to 39 kb; and class E was 29 kb. All strains contained at least two plasmids in classes A and B. The conjugative ability of P. syringae pv. tomato plasmids in three strains was demonstrated by mobilization of the nonconjugative plasmid RSF1010 into Pseudomonas syringae pv. syringae recipients. Plasmids from the three conjugative strains were labeled with Tn5. Four conjugative plasmids were identified by their repeated transfer to P. syringae pv. syringae recipients. P. syringae pv. tomato strains varied in sensitivity to copper sulfate (CuSO4): MICs were 0.4 to 0.6 mM for sensitive strains, 1.2 mM for moderately resistant strains, and 1.6 to 2.0 mM for very resistant strains. One very resistant strain, PT23, functioned as a donor of copper resistance. Recipient P. syringae pv. syringae strains PS51 and PS61 were inhibited by 0.1 mM CuSO4, whereas the CuSO4 MICs for transconjugant strains PS51(pPT23A) and PS61(pPT23C) were 1.8 and 2.6 mM, respectively. P. syringae pv. tomato strains PT12.2 and PT17.2 were inhibited by 0.6 mM copper sulfate, but their copper sulfate MICs were 2.6 and 1.8 mM, respectively, when they acquired pPT23C. Therefore, copper resistance in PT23 was controlled by two conjugative plasmids, designated pPT23A (101 kb) and pPT23C (67 kb).     

Plasmid-Determined Copper Resistance in Pseudomonas syringae from Impatiens

A strain of Pseudomonas syringae was recently identified as the cause of a new foliar blight of impatiens. The bacterium was resistant to copper compounds, which are used on a variety of crops for bacterial and fungal disease control. The bacterium contained a single 47-kilobase plasmid (pPSI1) that showed homology to a copper resistance operon previously cloned and characterized from P. syringae pv. tomato plasmid pPT23D (D. Cooksey, Appl. Environ. Microbiol. 53:454-456, 1987). pPSI1 was transformed by electroporation into a copper-sensitive P. syringae strain, and the resulting transformants were copper resistant. A physical map of pPSI1 was constructed, and the extent of homology to pPT23D outside the copper resistance operon was determined in Southern hybridizations. The two plasmids shared approximately 20 kilobases of homologous DNA, with the remainder of each plasmid showing no detectable homology. The homologous regions hybridized strongly, but there was little or no conservation of restriction enzyme recognition sites.     

Early detection of bean infection by Pseudomonas syringae in asymptomatic leaf areas using chlorophyll fluorescence imaging

Chlorophyll fluorescence imaging has been used to analyse the response elicited in Phaseolus vulgaris after inoculation with Pseudomonas syringae pv. phaseolicola 1448A (compatible interaction) and P. syringae pv. tomato DC3000 (incompatible interaction). With the aim of modulating timing of symptom development, different cell densities were used to inoculate bean plants and the population dynamics of both bacterial strains was followed within the leaf tissue. Fluorescence quenching analysis was carried out and images of the different chlorophyll fluorescence parameters were obtained for infected as well as control plants at different timepoints post-infection. Among the different parameters analysed, we observed that non-photochemical quenching maximised the differences between the compatible and the incompatible interaction before the appearance of visual symptom. A decrease in non-photochemical quenching, evident in both infiltrated and non-infiltrated leaf areas, was observed in P. syringae pv. phaseolicola-infected plants as compared with corresponding values from controls and P. syringae pv. tomato-infected plants. No photoinhibitory damage was detected, as the maximum photosystem II quantum yield remained stable during the infection period analysed.     

Polysaccharides of <Emphasis Type="Italic">Pseudomonas</Emphasis> Pathovar Strains That Infect Pea,Tomato, and Soya Bean

In order to understand the mode of action of taxonomically related Pseudomonas syringae pathovar strains that infect pea, tomato, and soya bean, we examined their extracellular polysaccharides (EPS). Maximum production of polysaccharide in shake culture of these pathogens was observed between 24 and 60 h. P. syringae pv. pisi 519, the bacterial blight pathogen of pea, produced a higher amount of polysaccharide (34.87 g/mL) at 60 h compared with 32.67 g/mL produced by P. syringae pv. glycinea NCPPB 1783, the bacterial blight pathogen of soya bean, and 30.03 g/mL produced by P. syringae pv. tomato NCPPB 269, the bacterial speck pathogen of tomato. EPS produced by P. syringae pv. pisi 519, P. syringae pv. tomato NCPPB 269, and P. syringae pv. glycinea NCPPB 1783 was characterized with infrared (FTIR), nuclear magnetic resonance (NMR), high performance thin layer chromatography, (HPTLC), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. HPTLC profiles revealed the presence of glucose and glucuronic acid in all bacteria and mannose only in P. syringae pv. tomato. Molecular mass of EPS of P. syringae pv. pisi (m/z 933.8), P. syringae pv. tomato (m/z 950.4), and P. syringae pv. glycinea (m/z 933.5) was confirmed by MALDI-TOF mass spectrometry.     

Inheritance and sources of resistance to bacterial speck of tomato caused by Pseudomonas syringae pv. tomato

Inheritance of resistance to bacterial speck of tomato was determined by analysing F₁ F₂ and backcross progenies of crosses involving a susceptible (VF-198) and a resistant cultivar (Rehovot-13). The results fit the hypothesis that resistance is controlled by a single dominant gene in interaction with minor genes. Cultivar susceptibility to Pseudomonas syringae pv. tomato was tested under greenhouse conditions under high inoculum pressure using infested tomato seeds together with infested soils and spray-inoculated wounded plants. Of 21 species, cultivars and lines, Rehovot-13, Ontario 7710 and Lycopersiconpimpinellifolium P.I. 126927 were found to be resistant to the pathogen. VF-198 and Tropic-VF were the most susceptible. Extra Marmande, Saladette, Acc.339944–3 and the wild type Lycopersicon esculentum var. cerasiforme were moderately resistant.     

Similarity between Copper Resistance Genes from Xanthomonas campestris and Pseudomonas syringae

Plasmid-borne copper resistance genes from copper-resistant strains of Xanthomonas campestris pv. vesicatoria from California, Florida, and Oklahoma shared structural similarities. A strain of X. campestris pv. campestris also contained plasmid-borne copper resistance genes similar to the resistance genes from X. campestris pv. vesicatoria. Furthermore, a region of the copper resistance genes from X. campestris pv. vesicatoria 07882 hybridized with copA, the first gene of the copper resistance operon (cop) of Pseudomonas syringae pv. tomato. A copper-inducible protein of similar size to CopA was detected by Western blot (immunoblot) analysis from the wild-type strain 07882 and from the cloned copper resistance genes of 07882 introduced into a copper-sensitive strain of X. campestris pv. vesicatoria. A low level of hybridization was observed with chromosomal DNA from other xanthomonads when the copper resistance genes from strain 07882 were used as probes.     

Antibacterial Activity of Tea and Coffee Wastes Against Some Plant Pathogenic Pseudomonas syringae Strains

In vitro and greenhouse trials were conducted to elucidate the potential use of extracts of tea and coffee wastes to control plant diseases caused by the bacterial pathogens, Pseudomonas syringae pv. pisi (race 1 and 2) and P. s. pv. phaseolicola (race 1). The antibacterial activity was measured as the diameter of the inhibition zone in agar and also by periodical viable cell counts in laboratory tests. The effect on the hypersensitive reaction and the potential for disease control after leaf infiltration and seed treatment were studied on bean plants in the greenhouse. Results showed that both the tea and coffee extracts possessed antibacterial activity against the three pathogens, but that the effects varied depending on the strain and the test method. Strong reduction of halo blight disease and improvement in plant growth was obtained in presence of the coffee extract. For the halo blight pathogen, P. s. pv. phaseolicola, there was a good correlation between the results from the viable cell count method and the greenhouse tests, but the results from the in vitro studies did not agree with those from greenhouse as regards the P. s. pv. pisi strains. It is suggested that the component(s) in tea and coffee responsible for controlling the bean pathogen may not be the same as that for the pea pathogens.     

Ecological and genetic analysis of copper and streptomycin resistance in Pseudomonas syringae pv. syringae.

Strains of Pseudomonas syringae pv. syringae resistant to copper, streptomycin, or both compounds were recovered from symptomless and diseased tissue of four woody hosts in three nurseries in Oklahoma. In strains resistant to copper and streptomycin (Cur Smr), resistance to both compounds was cotransferred with a single plasmid which was either 68, 190, or 220 kilobase pairs (kb). All Cus Smr strains contained a 68-kb conjugative plasmid. Cur Sms strains contained one plasmid which varied in size from 60 to 73 kb. All conjugative plasmids which transferred streptomycin resistance contained sequences homologous to the strA and strB Smr genes from the broad-host-range plasmid RSF1010. The Smr determinant was subsequently cloned from a 68-kb Cur Smr plasmid designated pPSR1. A restriction map detailing the organization of the homologous Smr genes from pPSR1 and RSF1010 and cloned Smr genes from P. syringae pv. papulans and Xanthomonas campestris pv. vesicatoria revealed the conservation of all sites studied. The Cur genes cloned from P. syringae pv. tomato PT23 and X. campestris pv. vesicatoria XV10 did not hybridize to the Cur plasmids identified in the present study, indicating that copper resistance in these P. syringae pv. syringae strains may be conferred by a distinct genetic determinant.     

Rapid linkage of indole carboxylic acid to the plant cell wall identified as a component of basal defence in Arabidopsis against hrp mutant bacteria

Changes occurring to plant cell walls were examined following inoculation of Arabidopsis leaves with pathogenic and non-pathogenic (hrpA mutant) strains of Pseudomonas syringae pv. tomato. We have targeted low molecular weight, cross-linked phenolic and indolic compounds that were released from wall preparations by alkaline hydrolysis at 70 °C and in a microwave bomb. Significantly higher concentrations of syringaldehyde, p hydroxybenzaldehyde and indole carboxylic acid were recovered from cell walls isolated from leaves 24 h after challenge with the hrpA mutant compared with wild-type DC3000. Time course experiments showed that the accumulation of indole carboxylic acid and the other group of differentiating metabolites had occurred within 12 h of inoculation. The callose synthase deficient mutant pmr4-1 was more resistant than wild-type Columbia plants to P. syringae pv. tomato. Restricted bacterial multiplication was associated with increased accumulation of indole carboxylic acid on the plant cell wall. In the absence of callose deposition in the pmr 4-1 mutant, indolic derivatives may serve as a structural scaffold for wall modifications following bacterial challenge.     

<Emphasis Type="Italic">PCC1</Emphasis>: a merging point for pathogen defence and circadian signalling in<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Using a cDNA-array we identified expressed sequence tag 163B24T7 as rapidly up-regulated in Arabidopsis thaliana (L.) Heynh. after pathogen exposure. Detailed expression analysis revealed that the corresponding gene is up-regulated not only after exposure to avirulent Pseudomonas syringae pv. tomato but also to virulent strains. This up-regulation is dependent on functional salicylic acid defence-signalling pathways. Moreover, we found the gene was circadian-regulated, showing peaks of expression at the end of the day. Using plants overexpressing the clock component CCA1, we showed that the PCC1 gene is regulated by the inner clock of Arabidopsis. Accordingly, we named the gene PCC1, for pathogen and circadian controlled. PCC1 is a member of a novel family of six small polypeptides in Arabidopsis. A functional role for PCC1 in plant defence was demonstrated since plants overexpressing PCC1 are resistant against normally virulent oomycetes. Thus, PCC1 demonstrates a potential interrelationship between pathogen and circadian signalling pathways.Abbreviations cfu Colony-forming units - EST Expressed sequence tag - Pst Pseudomonas syringae pv. tomato - TAIR The Arabidopsis information resource     

The improvement of resistance to bacterial speck in transgenic tomato plants by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> mediated transformation

The pto gene, responsible for resistance to Pseudomonas syringae pv. tomato, was transferred to tomato genotype Urfa-2 by the LBA4404 strain of A. tumefaciens harboring the plasmid pPTC8. The presence of nptII and pto genes in transgenic plants was proved by PCR analysis. Insertion of the pto gene into the genome of transgenic plants and expression of the gene were confirmed by southern and northern hybridizations, respectively. The pathogen P. syringae pv. tomato was applied to all leaves of transgenic and control plants. While typical bacterial speck symptoms developed on the leaves of control plants, the transgenic plants did not display any typical symptoms of bacterial speck upon inoculation with strains 1 and 0. Some of these transgenic plants had thicker leaves than the control plants and produced abnormal flowers. The pollen of transgenic plants was used for crossing with control plants to produce F1 transgenic lines. Fruits from crossed transgenic and control plants were obtained, and F1 seeds germinated on Murashige and Skoog medium in the presence of kanamycin have developed F1 seedlings. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 1, pp. 102–110. The text was submitted by the authors in English.