Characterisation of basal resistance (BR) by expression patterns of newly isolated representative genes in tobacco

Increasing evidence indicates that plants, like animals, use basal resistance (BR), a component of the innate immune system, to defend themselves against foreign organisms. Contrary to the hypersensitive reaction (HR)-type cell death, recognition in the case of BR is unspecific, as intruders are recognised based on their common molecular patterns. Induction of BR is not associated with visible symptoms, in contrast to the HR-type cell death. To analyse the early events of BR in tobacco plants we have carried out a subtractive hybridisation between leaves treated with the HR-negative mutant strain Pseudomonas syringae pv. syringae 61 hrcC and non-treated control leaves. Random sequencing from the 304 EBR clones yielded 20 unique EST-s. Real-time PCR has proved that 8 out of 10 clones are activated during BR. Six of these EST-s were further analyzed. Gene expression patterns in a time course showed early peaks of most selected genes at 3–12 h after inoculation (hpi), which coincided with the development-time of BR. Upon treatments with different types of bacteria we found that incompatible pathogens, their hrp mutants, as well as non-pathogens induce high levels of expression while virulent pathogens induce only a limited gene-expression. Plant signal molecules like salicylic acid, methyl jasmonate, ethylene and spermine, known to be involved in plant defense were not able to induce the investigated genes, therefore, an unknown signalling mechanism is expected to operate in BR. In summary, we have identified representative genes associated with BR and have established important features of BR by analysing gene-expression patterns.     

Gac two-component system in Pseudomonas syringae pv. tabaci is required for virulence but not for hypersensitive reaction

Pseudomonas syringae pv. tabaci 6605 causes wildfire disease on host tobacco plants. To investigate the regulatory mechanism of the expression of virulence, Gac two-component system-defective mutants, ΔgacA and ΔgacS, and a double mutant, ΔgacAΔgacS, were generated. These mutants produced smaller amounts of N-acyl homoserine lactones required for quorum sensing, had lost swarming motility, and had reduced expression of virulence-related hrp genes and the algT gene required for exopolysaccharide production. The ability of the mutants to cause disease symptoms in their host tobacco plant was remarkably reduced, while they retained the ability to induce hypersensitive reaction (HR) in the nonhost plants. These results indicated that the Gac two-component system of P. syringae pv. tabaci 6605 is indispensable for virulence on the host plant, but not for HR induction in the nonhost plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequence data reported in this paper have been submitted to the DDBJ/GenBank/EMBL databank with the accession numbers AB266103, AB266104, AB266105, AB266106, AB266107, AB266108.     

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.     

Comparative analysis of induction pattern of programmed cell death and defense-related responses during hypersensitive cell death and development of bacterial necrotic leaf spots in eggplant

Pseudomonas cichorii causes necrotic leaf spots (NLS), while Pseudomonas syringae pv. tabaci induces a hypersensitive response (HR) in eggplant. P. cichorii induced cell death at 9 h after inoculation (HAI), reaching a maximum of around 24–30 HAI. On the other hand, cell death was induced 6 HAI with P. syringae pv. tabaci, reaching a maximum of around 12–18 HAI. Superoxide generation was observed in eggplant inoculated with both bacteria. DNA fragmentation, cytochrome c release into the cytosol and expression of defense-related genes such as PR-1 and hsr203J was also induced by inoculation with both bacteria, but these plant reactions were more rapidly induced in eggplant inoculated with P. syringae pv. tabaci rather than those with P. cichorii. Lipid peroxidation and induction of lipoxygenase (LOX) was drastically induced in eggplant inoculated with P. syringae pv. tabaci compared to P. cichorii-inoculated eggplant. Pharmacological studies showed that induction of the cell death, and the NLS or the HR in response to both bacteria was commonly associated with de novo protein synthesis, reactive oxygen species and caspase III-like protease. Interestingly, involvement of lipid peroxidation, LOX, serine protease, and DNase differed between induction of NLS and HR. These results suggest that programmed cell death might be closely associated not only with the HR but also NLS. However, there may be differences not only in the induction kinetics and level of plant responses but also in the infection-related responses between HR and NLS.     

Cloning of genes required for hypersensitivity and pathogenicity inPseudomonas syringae pv.aptata

A genomic library ofPseudomonas syringae pv.aptata strain NCPPB 2664, which causes bacterial blight of sugar beet, lettuce and other plants, was constructed in the cosmid vector pCPP31. The 13.4 kbEcoRI fragment of the cosmid pHIR11, containing thehrp (hypersensitiveresponse andpathogenicity) gene cluster of the closely related bacteriumPseudomonas syringae pv.syringae strain 61, was used as a probe to identify a homologoushrp gene cluster inP. syringae pv.aptata. Thirty of 2500 cosmid clones, screened by colony hybridization, gave a strong hybridization signal with the probe, but none of these conferred to the non-pathogenic bacterium,Pseudomonas fluorescens, the ability to elicit the hypersensitive response (HR) in tobacco. Southern blot analysis ofEcoRI-digested genomic DNA ofP. syringae pv.aptata showed hybridizing bands of 12 kb and 4.4 kb. Only a 12 kb fragment hybridized in digests of the cosmids. Cosmid clone pCPP1069 was mutagenized with Tn10-minitet and marker-exchanged into the genome ofP. syringae pv.aptata. Three resulting prototrophic mutant strains failed to elicit the HR in tobacco and to cause disease in lettuce. The DNA flanking the Tn10-minitet insertions from mutated derivatives of pCPP1069 hybridized with the 10.6 kbBglII fragment of pHIR11. These results indicate thatP. syringae pv.aptata harbourshrp genes that are similar to, but arranged differently from, homologoushrp genes ofP. syringae pv.syringae.Abbreviations HR hypersensitive response - Hrp mutant unable to induce HR and pathogenicity - Psa Pseudomonas syringae pv.aptata - Pss Pseudomonas syringae pv.syringae - Ea Erwinia amylovora     

The genetic characterization of Pseudomonas syringae pv. tagetis based on the 16S–23S rDNA intergenic spacer regions

Pseudomonas syringae pv. tagetis, a plant pathogen being considered as a biological control agent of Canada thistle (Cirsium arvense), produces tagetitoxin, an inhibitor of RNA polymerase which results in chlorosis of developing shoot tissues. Although the bacterium is known to affect several plant species in the Asteraceae and has been reported in several countries, little is known of its genetic diversity. The genetic relatedness of 24 strains of P. syringae pv. tagetis with respect to each other and to other P. syringae and Pseudomonas savastanoi pathovars was examined using 16S–23S rDNA intergenic spacer (ITS) sequence analysis. The size of the 16S–23S rDNA ITS regions ranged from 508 to 548 bp in length for all 17 P. syringae and P. savastanoi pathovars examined. The size of the 16S–23S rDNA ITS regions for all the P. syringae pv. helianthi and all the P. syringae pv. tagetis strains examined were 526 bp in length. Furthermore, the 16S–23S rDNA ITS regions of both P. syringae pv. tagetis and P. syringae pv. helianthi had DNA signatures at specific nucleotides that distinguished them from the 15 other P. syringae and P. savastanoi pathovars examined. These results provide strong evidence that P. syringae pv. helianthi is a nontoxigenic form of P. syringae pv. tagetis. The results also demonstrated that there is little genetic diversity among the known strains of P. syringae pv. tagetis. The genetic differences that do exist were not correlated with differences in host plant, geographical origin, or the ability to produce toxin.     

Insight into Types I and II nonhost resistance using expression patterns of defense-related genes in tobacco

Plants protect themselves against pathogens using a range of response mechanisms. There are two categories of nonhost resistance: Type I, which does not result in visible cell death; and Type II, which entails localized programmed cell death (or hypersensitive response) in response to nonhost pathogens. The genes responsible for these two systems have not yet been intensively investigated at the molecular level. Using tobacco plants (Nicotiana tabacum), we compared expression of 12 defense-related genes between a Type I (Xanthomonas axonopodis pv. glycines 8ra) nonhost interaction, and two Type II (Pseudomonas syringae pv. syringae 61 and P. syringae pv. phaseolicola NPS3121) nonhost interactions, as well as those expressed during R gene-mediated resistance to Tobacco mosaic virus. In general, expression of most defense-related genes during R gene-mediated resistance was activated 48 h after challenge by TMV; the same genes were upregulated as early as 9 h after infiltration by nonhost pathogens. Surprisingly, X. axonopodis pv. glycines (Type I) elicited the same set of defense-related genes as did two pathovars of P. syringae, despite the absence of visible cell death. In two examples of Type II nonhost interactions, P. syringae pv. phaseolicola NPS3121 produced an expression profile more closely resembling that of X. axonopodis pv. glycines 8ra, than that of P. syringae pv. syringae 61. These results suggest that Type I nonhost resistance may act as a mechanism providing a more specific and active defense response against a broad range of potential pathogens.     

BAHD superfamily of acyl-CoA dependent acyltransferases in <Emphasis Type="Italic">Populus</Emphasis> and <Emphasis Type="Italic">Arabidopsis</Emphasis>: bioinformatics and gene expression

Plant acyl-CoA dependent acyltransferases constitute a large specific protein superfamily, named BAHD. Using the conserved sequence motifs of BAHD members, we searched the genome sequences of Populus and Arabidopsis, and identified, respectively, 94- and 61-putative genes. Subsequently, we analyzed the phylogeny, gene structure, and chromosomal distribution of BAHD members of both species; then, we profiled expression patterns of BAHD genes by “in silico” northern- and microarray-analyses based on public databases, and by RT-PCR. While our genomic- and bioinformatic- analyses provided full sets of BAHD superfamily genes, and cleaned up a few existing annotation errors, importantly it led to our recognizing several unique Arabidopsis BAHD genes that inversely overlapped with their neighboring genes on the genome, and disclosing a potential natural anti-sense regulation for gene expressions. Systemic gene-expression profiling of BAHD members revealed distinct tissue-specific/preferential expression patterns, indicating their diverse biological functions. Our study affords a strong knowledge base for understanding BAHD members’ evolutionary relationships and gene functions implicated in plant growth, development and metabolism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Community analysis of betaproteobacterial ammonia-oxidizing bacteria using the <Emphasis Type="Italic">amoCAB</Emphasis> operon

The genes and intergenic regions of the amoCAB operon were analyzed to establish their potential as molecular markers for analyzing ammonia-oxidizing betaproteobacterial (beta-AOB) communities. Initially, sequence similarity for related taxa, evolutionary rates from linear regressions, and the presence of conserved and variable regions were analyzed for all available sequences of the complete amoCAB operon. The gene amoB showed the highest sequence variability of the three amo genes, suggesting that it might be a better molecular marker than the most frequently used amoA to resolve closely related AOB species. To test the suitability of using the amoCAB genes for community studies, a strategy involving nested PCR was employed. Primers to amplify the whole amoCAB operon and each individual gene were tested. The specificity of the products generated was analyzed by denaturing gradient gel electrophoresis, cloning, and sequencing. The fragments obtained showed different grades of sequence identity to amoCAB sequences in the GenBank database. The nested PCR approach provides a possibility to increase the sensitivity of detection of amo genes in samples with low abundance of AOB. It also allows the amplification of the almost complete amoA gene, with about 300 bp more sequence information than the previous approaches. The coupled study of all three amo genes and the intergenic spacer regions that are under different selection pressure might allow a more detailed analysis of the evolutionary processes, which are responsible for the differentiation of AOB communities in different habitats. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Pilar Junier and Ok-Sun Kim contributed equally to this work.     

<Emphasis Type="Italic">SQUA</Emphasis>-like genes in the orchid <Emphasis Type="Italic">Phalaenopsis</Emphasis> are expressed in both vegetative and reproductive tissues

The SQUA family (AP1/FUL family) of MADS-box genes plays an important role in the transition from the vegetative to the reproductive development of angiosperms, and its origin might be concurrent with fixation of floral structure in angiosperms. Here, we isolated two Phalaenopsis MADS-box genes designated ORAP11 and ORAP13, both of which belong to the monocot FUL-like clade of the SQUA family. RT-PCR showed that both genes are strongly expressed in the floral bud, and also detected in the vegetative organs. During later stages, ORAP11 was only detected in the column, but ORAP13 signal was absent from all of the floral organs. In-situ hybridization experiments detected both genes in the tips and margins of developing petals and lips, the developing column, and ovule. Over-expression of both genes in tobacco induced early flowering and changed plant architecture. Our results suggest that in Phalaenopsis, both genes might share partly redundant activities and play important roles in the process of floral transition and morphological architecture. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Phenotypic,genomic and phylogenetic characteristics of rhizobia isolated from root nodules of <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> (black locust) growing in Poland and Japan

Rhizobial strains, rescued from the root nodules of Robinia pseudoacacia growing in Japan and Poland, were characterized for the phenotypic properties, genomic diversity as well as phylogeny and compared with the reference strains representing different species and genera of nodule bacteria. They had a moderately slow growth rate, a low tolerance to antibiotics, a moderate resistance to NaCl and produced acid in yeast mannitol agar. Cluster analysis based on the phenotypic features divided all bacteria involved in this study into four phena, comprising: (1) Rhizobium sp. + Sinorhizobium sp., (2) Bradyrhizobium sp., (3) R. pseudoacacia microsymbionts + Mesorhizobium sp., and (4) Rhizobium galegae strains at similarity coefficient of 74%. R. pseudoacacia nodule isolates and Mesorhizobium species were placed on a single branch clearly distinct from other rhizobium genera lineages. Strains representing R. pseudoacacia microsymbionts shared 98–99% 16S rDNA sequence identity with Mesorhizobium species and in 16S rDNA phylogenetic tree all these bacteria formed common cluster. The rhizobia tested are genomically heterogeneous as indicated by the AFLP (Amplified Fragment Length Polymorphism) method. The bacteria studied exhibited high degree of specificity for nodulation. Nitrogenase structural genes in these strains were located on 771–961 kb megaplasmids. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">WUS</Emphasis> and <Emphasis Type="Italic">STM</Emphasis>-based reporter genes for studying meristem development in poplar

We describe the development of a reporter system for monitoring meristem initiation in poplar using promoters of poplar homologs to the meristem-active regulatory genes WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM). When ~3 kb of the 5′ flanking regions of close homologs were used to drive expression of the GUSPlus gene, 50–60% of the transgenic events showed expression in apical and axillary meristems. However, expression was also common in other organs, including in leaf veins (40 and 46% of WUS and STM transgenic events, respectively) and hydathodes (56% of WUS transgenic events). Histochemical GUS staining of explants during callogenesis and shoot regeneration using in vitro stems as explants showed that expression was detectable prior to visible shoot development, starting 3–15 days after explants were placed onto callus inducing medium. A minority of WUS and STM events also showed expression in the cambium, phloem, or xylem of regenerated, greenhouse grown plants undergoing secondary growth. Based on microarray gene expression data, a paralog of poplar WUS was detectably up-regulated during shoot initiation, but the other paralog was not. Both paralogs of poplar STM were down-regulated threefold to sixfold during early callus initiation. We identified 15–35 copies of cytokinin response regulator binding motifs (ARR1AT) and one copy of the auxin response element (AuxRE) in both promoters. Several of the events recovered may be useful for studying the process of primary and secondary meristem development, including treatments intended to stimulate meristem development to promote clonal propagation and genetic transformation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.