Molecular Plant-Microbe Interactions

Published by American Phytopathological Society
Extracellular and intracellular neutral beta-1,2-linked D-glucan content was determined in a virulent, attachment-deficient mutants of Agrobacterium tumefaciens that map in the chvA locus. chvA mutants contained approximately the same amount of intracellular glucan as cells of the virulent control strain A759, but released into the culture medium only 2% of the glucan released by strain A759. Introduction of a cosmid carrying the wild-type chv region restored attachment and virulence and restored extracellular glucan production to chvA mutant A2505. Exogenous glucan did not enhance or inhibit attachment or tumorigenesis of the virulent control strain or the chvA or chvB mutants. Our results suggest that the chvA locus is involved in the export of glucan from the cell and that export may be required for tumorigenesis.
Confocal laser scanning micrography of leaf section from AtBG2-green fluorescent protein (GFP)–expressing transgenic Nicotiana benthamiana infected with Tobacco mosaic virus, which lacks coat protein (CP) and expresses movement protein (MP) fused to red fluorescent protein (RFP) (TMV ΔCP - MP-RFP), at 4 days postinoculation. Infection stages indicated by I, II, and III correspond to zones of the infection ring. A to C, AtBG2-GFP and MP-RFP co-localize in virus-induced endoplasmic reticulum bodies in newly infected cells within zone I. The empty arrow in A indicates the direction of virus movement . Cell 0 is the cell in which MP-RFP is as-yet nondetectable. Cell 1 is the first cell in which MP-RFP accumulation can be detected. A, Enlargement of the marked area at the wall region (w) in cell 1 shows early formation of MP-RFP/AtBG2-GFP bodies at the cell periphery (arrows). Enlargement of the marked area at the upper cortical focus (c) in cell 1 shows the cortical MP-RFP/AtBG2-GFP body (arrowhead). D to F, AtBG2-GFP and MP-RFP localization in cells within zone II. G to I, AtBG2-GFP and MP-RFP localization in cells within zone III. G to I, Insert shows enlargement of the marked area at the upper cortical focus. A, D, and G, MP-RFP red channel only. B, E, and H, AtBG2-GFP green channel only. C, F, and I, Both green and red channels overlaid . Images in A to C are overlays with differential interference contrast channel. Bars: A to I = 10 μm; enlargements in A to C and G to I = 5 μm.  
Confocal laser-scanning micrography showing localization of movement protein fused to red fluorescent protein (MP-RFP), AtBG2-green fluorescent protein (GFP), and callose in cells at the leading edge (zone I) of infection with Tobacco mosaic virus (TMV), which lacks coat protein (CP) and expresses movement protein fused to red fluorescent protein (TMV Δ CP -MP-RFP). Transgenic Nicotiana benthamiana overexpressing AtBG2-GFP was infected with viral RNA and, at 4 days postinoculation, wound callose at plasmodesmata (Pd) (shown in white) was induced by cutting through the infection site followed by aniline blue staining. A to C, In initially infected cell, MP-RFP accumulates in small endoplasmic reticulum bodies appressed to the cell wall, often in the vicinity of Pd (arrows), and co-localizes with AtBG2-GFP. Cell 0 is the one in which MP-RFP is not yet detectable. Cell 1 is the first cell in which MP-RFP accumulation can be detected. D to F, Later, bodies appear as double fluorescent spots on both sides of Pd (arrow). G to I, Later yet, the bodies enlarge, fuse, and still flank Pd. A, D, and G, Overlays of MP-RFP red and callose white (pseudo-colored) channels. B, E, and H, Overlays of AtBG2-GFP green and callose white channels. C, F, and I, Three channels overlaid. G to I, Inserts show enlargements of the area marked by arrows with the callose channel turned off. All arrows indicate also the direction of virus spread. Bars: A to C and G to I = 10 μm and D to F = 5 μm.  
A and B, Confocal laser-scanning micrography showing localization of movement protein fused to red fluorescent protein (MP-RFP) and AtBG2- green fluorescent protein (GFP) in plasmolyzed leaf tissue infected with Tobacco mosaic virus (TMV), which lacks coat protein (CP) and expresses MP-RFP (TMV Δ CP -MP-RFP). A, In cells at the infection edge (zone I), AtBG2-GFP is associated with virus-induced endoplasmic reticulum (ER) bodies (yellow) but is not detected in the extracellular spaces formed between the retracted protoplast and cell wall. Cell 0 is the one in which MP-RFP is not yet detectable. Cell 1 is the first cell in which MP-RFP accumulation can be seen. B, In cells at the late stage of infection (zone III), AtBG2-GFP accumulates in ER, does not co-localize with MP, and is absent from the formed extracellular spaces. Asterisks in A and B indicate the extracellular spaces formed after plasmolysis and arrowheads point out the retracted plasma membrane. C to H, Localization of AtBG2-GFP and GFP-HDEL (GFP with an ER retention signal) in cells at the infection front (zone I) following salicylic acid (SA) treatment. Infected leaves expressing either AtBG2-GFP or GFP-HDEL were detached at 3 days postinoculation , and the petiole was placed either in water (–SA) or 2.5 mM SA (+SA) for 24 h. C, Water-treated infection site has a larger ring and small central zone. MP-RFP co-localizes with both D, AtBG2-GFP and E, GFP-HDEL in small ER-derived bodies at the infection edge. F, SA-treated infection site has a smaller ring with enraged central zone. G and H, At the infection edge, the MP-RFP ER bodies are large, characteristic of later stage of infection. G, Most of the AtBG2-GFP is secreted out of the ER bodies (red) and appear as continuous green fluorescence in the cell wall (arrow). H, SA has no effect on GFP- HDEL localization. Bars: A, B, D, E, G, and H = 10 μm and C and F = 500 μm.  
A, Callose accumulation at plasmodesmata (Pd) and B, local spread of TVCV-green fluorescent protein (GFP) in wild-type (WT) and two knockout Arabidopsis β-1,3-glucanase (BG) mutants, atbg_ pap and atbg2. A, Callose levels at Pd in atbg_ pap and atbg2 mutants relative to WT plants. The representative images of aniline-blue-stained leaf epidermis are shown in upper panels. For each line, three plants and three leaves on each plant were used for callose measurements. At least 30 images for each line were analyzed. Values are presented as percentage of average of callose intensity from Pd sites in mutants relative to that in WT plants ± standard error (SE). Asterisk indicates statistical difference of the mean at P < 0.0001, as determined by Student's t test. The experiment was repeated twice, with similar results. B, Local spread of TVCV-GFP in WT, atbg_ pap, and atbg2 mutants quantified by measuring the area of fluorescent infection sites at 3.5 to 7.5 days postinoculation (dpi) from the inoculated adaxial leaf surface. The representative fluorescent infection sites at 6.5 dpi are shown in upper panels. Two plants and three leaves on each plant were inoculated for each time point. Data are presented as the average area of infection sites ± SE. Lowercase letters indicate statistical differences of the mean at P < 0.001, as determined by Student's t test for each time point. The experiment was repeated twice with similar results. Numbers within columns indicate number of infection sites measured (n). Bars: A = 20 μm and B = 1 mm.  
β-1,3-glucanases (BGs) have been implicated in enhancing virus spread by degrading callose at plasmodesmata (Pd). Here we investigate the role of Arabidopsis BGs in tobamovirus spread. During Turnip vein clearing virus infection the transcription of two pathogenesis related BGs (PR-BGs), AtBG2 and AtBG3, increased, but that of Pd-associated BG, AtBG_pap, did not change. In transgenic plants AtBG2 was retained in the endoplasmic reticulum (ER) network and was not secreted. As a stress response mediated by salicylic acid AtBG2 was secreted and appeared as a free extracellular protein localized in the entire apoplast, but did not accumulate at Pd sites. At the leading edge of Tobacco mosaic virus spread, AtBG2 co-localized with the viral movement protein in the ER-derived bodies, similarly to other ER proteins, but was not secreted to cell wall. In atbg2 mutants callose levels at Pd and virus spread were unaffected. AtBG2 over-expression had no effect on virus spread as well. However, in atbg_pap mutants callose at Pd was increased and virus spread was reduced. Our results demonstrate that the constitutive Pd-associated BGs, but not the stress regulated extracellular PR-BGs, are directly involved in regulation of callose at Pd and cell-to-cell transport in Arabidopsis, including the spread of viruses.
The modes of action of the antagonistic yeast Pichia anomala (strain K) have been studied; however, thus far, there has been no clear demonstration of the involvement of exo-beta-1,3-glucanase in determining the level of protection against Botrytis cinerea afforded by this biocontrol agent on apple. In the present study, the exo-beta-1,3-glucanase-encoding genes PAEXG1 and PAEXG2, previously sequenced from the strain K genome, were separately and sequentially disrupted. Transfer of the URA3-Blaster technique to strain K, allowing multiple use of URA3 marker gene, first was validated by efficient inactivation of the PaTRP1 gene and recovery of a double auxotrophic strain (uracil and tryptophan). The PAEXG1 and PAEXG2 genes then were inactivated separately and sequentially with the unique URA3 marker gene. The resulting mutant strains showed a significantly reduced efficiency of biocontrol of B. cinerea when applied to wounded apple fruit, the calculated protection level dropping from 71% (parental strain) to 8% (mutated strain) under some experimental conditions. This suggests that exo-beta-1,3-glucanases play a role in the biological control of B. cinerea on apple. Furthermore, biological control experiments carried out in this study underline the complexity of the host-antagonist-pathogen interaction. Two experimental parameters (yeast inoculum concentration and physiological stage of the fruit) were found to influence dramatically the protection level. Results also suggest that, under some conditions, the contribution of exo-beta-1,3-glucanase to biological control may be masked by other modes of action, such as competition.
An acidic beta-1,3-glucanase was detected in cucumber leaves inoculated with either Colletotrichum lagenarium or tobacco necrosis virus (TNV) as well as in the leaves above those inoculated with the pathogens. The enzyme is extracellular and migrates in native polyacrylamide gel electrophoresis (PAGE) together with a Class III chitinase, a bifunctional chitinase/lysozyme. The beta-1,3-glucanase was separated by ultra-narrow pH range IEF-PAGE or by SDS_PAGE and was purified to apparent homogeneity. Only one isoform of the enzyme was detected. Its apparent molecular mass in 38 kDa as estimated by SDS-PAGE, its isoelectric point is 3.6 and the specific activity is approximately 26 micromol glucose equivalents liberated from laminarin min(-1)mg(-1) protein. Partial amino acid (five peptide fragments with a total of 65 amino acids) sequencing of the beta-1,3-glucanase revealed similarities of 49% to 72% to sequences of published beta-1,3-glucanases from tobacco, tomato, soybean, barley, and rice plants. A time course study indicated that the increase of the beta-1,3-glucanase activity was associated with induced resistance against C. lagenarium. The implications of these results to coordinate defense responses in plant-microbe interactions are discussed.
Three pathogenesis-related (PR) proteins of tobacco are acidic isoforms of beta-1,3-glucanase (PR-2a, -2b, -2c). We have cloned and sequenced a partial cDNA clone (lambda FJ1) corresponding to one of the PR-2 beta-1,3-glucanases. A small gene family encodes the PR-2 proteins in tobacco, and similar genes are present in a number of plant species. We analyzed the stress and developmental regulation of the tobacco PR-2 beta-1,3-glucanases by using northern and western analyses and a new technique to assay enzymatic activity. Stress caused by both thiamine and tobacco mosaic virus (TMV) infection resulted in a dramatic increase in the levels of PR-2 mRNA, protein, and enzyme activities. The increased PR-2 gene expression in upper uninoculated leaves of plants infected with TMV also suggests a role in systemic acquired resistance. During floral development, a number of beta-1,3-glucanase activities were observed in all flower tissues. However, PR-2 polypeptides were observed only in sepal tissue. In contrast, an mRNA that hybridized to the PR-2 cDNA was present in stigma/style tissue and the sepals. Primer extension analysis confirmed the identity of the PR-2 mRNA in sepals, but indicated that the beta-1,3-glucanase gene expressed in the stigma/style of flowers was distinct from the PR-2 genes. The induction of PR-2 protein synthesis by both stress and developmental signals was accompanied by a corresponding increase in the steady-state levels of PR-2 mRNA, suggesting that PR-2 gene expression is regulated, in part, at the level of mRNA accumulation.
The sequence of a partial cDNA clone corresponding to an mRNA induced in leaves of barley (Hordeum vulgare) by infection with fungal pathogens matched almost perfectly with that of a cDNA clone coding for beta-1,-3-glucanase isolated from the scutellum of barley. Western blot analysis of intercellular proteins from near-isogenic barley lines inoculated with the powdery mildew fungus (Erysiphe graminis f. sp. hordei) showed a strong induction of glucanase in all inoculated lines but was most pronounced in two resistant lines. These data were confirmed by beta-1,3-glucanase assays. The barley cDNA was used as a hybridization probe to detect mRNAs in barley, wheat (Triticum aestivum), rice (oryza sativus), and sorghum (Sorghum bicolor), which are induced by infection with the necrotrophic pathogen Bipolaris sorokiniana. These results demonstrate that activation of beta-1,3-glucanase genes may be a general response of cereals to infection by fungal pathogens.
Results of codon evolution analyses of glucanase inhibitor proteins (GIPs) and serine protease homologs (SPH) homologs a P value b 
During invasion of their plant hosts, species of the oomycete genus Phytophthora secrete glucanase inhibitor proteins (GIPs) into the plant apoplast, which bind and inhibit the activity of plant extracellular endo-beta-1,3-glucanases (EGases). GIPs show structural homology to the chymotrypsin class of serine proteases (SP) but lack proteolytic activity due to the absence of an intact catalytic triad and, thus, belong to a broader class of proteins called serine protease homologs (SPH). To study the evolutionary relationship between GIPs and functional SP, database searches were used to identify 48 GIP homologs in the P. sojae, P. ramorum, and P. infestans genomes, composing GIPs, SPH, and potentially functional SP. Analyses of P. infestans-inoculated tomato leaves showed that P. infestans GIPs and tomato EGases are present in the apoplast and form stable complexes in planta. Studies of the temporal expression of a four-membered GIP family from P. infestans (PiGIP1 to PiGIP4) further revealed that the genes show distinctly different patterns during an infection timecourse. Codon evolution analyses of GIP homologs identified several positively selected peptide sites and structural modeling revealed them to be in close proximity to rapidly evolving EGase residues, suggesting that the interaction between GIPs and EGases has the hallmarks of a coevolving molecular arms race.
Sulfated laminarin (PS3) has been shown previously to be an elicitor of plant defense reactions in tobacco and Arabidopsis and to induce protection against tobacco mosaic virus. Here, we have demonstrated the efficiency of PS3 in protecting a susceptible grapevine cultivar (Vitis vinifera cv. Marselan) against downy mildew (Plasmopara viticola) under glasshouse conditions. This induced resistance was associated with potentiated H2O2 production at the infection sites, upregulation of defense-related genes, callose and phenol depositions, and hypersensitive response-like cell death. Interestingly, similar responses were observed following P. viticola inoculation in a tolerant grapevine hybrid cultivar (Solaris). A pharmacological approach led us to conclude that both callose synthesis and jasmonic acid pathway contribute to PS3-induced resistance.
Semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis of tomato endo-β-1,4-glucanase (EGase) transcripts in roots. RT-PCR products of seven tomato EGases were amplified from corresponding uninfected (U) and potato cyst nematode-infected (7 days postinoculation, I) root segments. Ubiquitin transcript levels were used to normalize the data.  
In situ detection of A, B, E, F, I, and J, Sl-cel7 and C, D, G, H, K, and L, Sl-cel9C1 transcripts on longitudinal sections of the susceptible tomato cv. Moneymaker roots infected with potato cyst nematode. Hybridization signal is visible as green coloration due to FITC fluorescence. Sections through uninfected roots hybridized with A, Sl-cel7 antisense probe; B, Sl-cel7 sense probe; C, Sl-cel9C1 antisense probe; and D, Sl-cel9C1 sense probe. Sections through syncytia (5 days postinoculation [dpi]) hybridized with E, Sl-cel7 antisense probe; F, Sl-cel7 sense probe; G, Sl-cel9C1 antisense probe; and H, Sl- cel9C1 sense probe. Sections through syncytia (10 dpi) hybridized with I, Sl-cel7 antisense probe; J, Sl-cel7 sense probe; K, Sl-cel9C1 antisense probe; and L, Sl-cel9C1 sense probe. N = nematode, Nu = nucleus, S = syncytium, and X = xylem. Bars: 30 μm.  
For the proliferation of their feeding sites (syncytia), the potato cyst nematode Globodera rostochiensis is thought to recruit plant endo-beta-1,4-glucanases (EGases, EC. Reverse-transcription polymerase chain reaction experiments on tomato (Solanum lycopersicum) indicated that the expression of two out of the at least eight EGases, namely Sl-cel7 and Sl-cel9C1, is specifically upregulated during syncytium formation. In situ hybridization and immunodetection studies demonstrated that both EGases are specifically expressed inside and adjacent to proliferating syncytia. To assess the importance of Sl-cel7 and Sl-cel9C1 for nematode development, we decided to knock them out individually. Sl-cel9C1 probably is the only class C EGase in tomato, and we were unable to regenerate Sl-cel9C1-silenced plants. Potato (S. tuberosum), a close relative of tomato, harbors at least two class C EGases, and St-cel7-or St-cel9C1-silenced potato plants showed no obvious aberrant phenotype. Infection with potato cyst nematodes resulted in a severe reduction of the number of adult females (up to 60%) and a sharp increase in the fraction of females without eggs (up to 89%). Hence, the recruitment of CEL7, an enzyme that uses xyloglucan and noncrystalline cellulose as natural substrates, and CEL9C1, an enzyme that uses crystalline cellulose, is essential for growth and development of potato cyst nematodes.
Western blot (immunoblots) of proteins from homogenates of second-stage juveniles of Heterodera glycines (lanes 1–4) and proteins from homogenates of noninoculated soybean roots (lanes 5–7) probed with antibodies to H. glycines (HG-ENG-1 and HG-ENG-2) β-1,4- endoglucanases. Lane 1: Pre-immune rabbit serum; lane 2: MGR 48 monoclonal antibody; lane 3: antiserum to HG-ENG-1 endoglucanase; lane 4: antiserum to HG-ENG-2 endoglucanase; lane 5: pre-immune rabbit serum; lane 6: antiserum to HG-ENG-1 endoglucanase; and lane 7: antiserum to HG-ENG-2 endoglucanase.  
Polyclonal sera specific to beta-1,4-endoglucanases (cellulases) synthesized in the subventral esophageal gland cells of the soybean cyst nematode, Heterodera glycines, were used to provide the first identification of a nematode esophageal gland protein that is secreted into host plant tissue. Sera generated to proteins encoded by Hg-eng-1 and Hg-eng-2 (endoglucanases) did not cross-react with soybean root proteins on Western blots (immunoblots) or in immunofluorescence microscopy of noninoculated (control) soybean root sections. In cross sections of soybean roots at 24 h after inoculation of roots with second-stage juveniles of H. glycines, HG-ENG-1 was localized within the nematode's subventral gland cells and was not detected in root tissue. HG-ENG-2 was localized within the subventral gland cells and was secreted from the juvenile's stylet into root cortical tissue at 24 h after inoculation of roots with second-stage juveniles of H. glycines. HG-ENG-2 was localized along the juvenile's migratory path through the root cortex.
Clones with secreted cellulolytic activity were identified when a cDNA library constructed from poly A(+) RNA of preparasitic second-stage juveniles of Heterodera glycines, the soybean cyst nematode, was expressed in the Escherichia coli SOLR strain and overlaid with a carboxymethylcellulose (CMC) substrate. Twenty CMC-degrading clones were analyzed, and all were either identical or strongly similar to a beta-1,4-endoglucanase gene (HG-eng-2), previously isolated from H. glycines. A subgroup of identical "HG-eng-2-like" clones had considerable differences in the 5' untranslated region compared with HG-eng-2 and were designated HG-eng-3. One H. glycines genomic clone contained HG-eng-2 and HG-eng-3 full-length genes, separated by a distance of approximately 8 kb, and a second genomic clone contained two copies of HG-eng-2, separated by approximately 6.5 kb, suggesting the presence of endoglucanase gene clusters in H. glycines. The HG-eng-2 and HG-eng-3 genes were in opposite transcriptional orientation, with considerable nucleotide differences in their 5' flanking regions. The highly conserved nucleotide sequence in the introns and exons and their close proximity within the genome suggest that HG-eng-2 and HG-eng-3 are the products of recent gene duplication and inversion.
Comparison of root length, root hair development, and root epidermal cell morphology of wild-type Arabidopsis thaliana ecotype Columbia (Col-0) and A. thaliana mutant rhd1-4 plants grown on normal medium and 2-aminoethoxyvinylglycine (AVG)-or 1-aminocyclo-propane-1-carboxylic acid (ACC)-supplemented medium. A, Macroscopic comparison of Col-0 and rhd1-4. Scale bar = 1,000 µm. B, Comparison of Col-0 and rhd1-4 root hair phenotypes. Scale bar = 400 µm. C, Comparison of root epidermal cell morphology of Col-0 and rhd1-4. Scale bar = 100 µm. D, Effect of 1.0 µM AVG on rhd1-4 root epidermis and root hairs (compare to C). Scale bar = 100 µm. E, Effects of the ethylene precursor ACC on root hair development in Col-0 and rhd1-4 (compare to B). Scale bars = 400 µm. 
Two beta-1,4-endoglucanases (EGases), Hg-eng-1 and Hg-eng-2, were recently cloned from the soybean cyst nematode, Heterodera glycines, and their expression was shown in the subventral esophageal glands of hatched second-stage juveniles (J2). We examined the expression of these EGases in the subventral glands of all post-embryonic life stages of H. glycines by in situ hybridization and immunolocalization. The first detectable accumulation of EGase mRNAs occurred in the subventral glands of unhatched J2. EGase transcripts remained detectable in J2 after hatching and during subsequent root invasion. However, in late parasitic J2 and third-stage juveniles (J3), the percentage of individuals that showed EGase transcripts decreased. In female fourth-stage juveniles and adult females, EGase transcripts were no longer detected in the subventral glands. EGase hybridization signal reappeared in unhatched males coiled within the J3 cuticle, and transcripts were also present in the subventral glands of migratory adult males. Immunofluorescence labeling showed that EGase translation products are most abundantly present in the subventral glands of preparasitic J2, migratory parasitic J2, and adult males. The presence of EGases predominantly in the migratory stages suggests that the enzymes are used by the nematodes to soften the walls of root cells during penetration and intracellular migration.
The phytopathogenic bacterium Clavibacter michiganensis subsp. michiganensis NCPPB382, which causes bacterial wilt and canker of tomato, harbors two plasmids, pCM1 (27.35 kb) and pCM2 (72 kb), encoding genes involved in virulence (D. Meletzus, A. Bermpohl, J. Dreier, and R. Eichenlaub, 1993, J. Bacteriol. 175:2131-2136; J. Dreier, D. Meletzus, and R. Eichenlaub, 1997, Mol. Plant-Microbe Interact. 10:195-206). The region of pCM1 carrying the endoglucanase gene celA was mapped by deletion analysis and complementation. RNA hybridization identified a 2.4-knt (kilonucleotide) transcript of the celA structural gene and the transcriptional initiation site was mapped. The celA gene encodes CelA, a protein of 78 kDa (746 amino acids) with similarity to endo-beta-1,4-glucanases of family A1 cellulases. CelA has a three-domain structure with a catalytic domain, a type IIa-like cellulose-binding domain, and a C-terminal domain. We present evidence that CelA plays a major role in pathogenicity, since wilt induction capability is obtained by endoglucanase expression in plasmid-free, nonvirulent strains and by complementation of the CelA- gene-replacement mutant CMM-H4 with the wild-type celA gene.
The gene, XYL1, encoding the major extracellular endo-beta 1,4-xylanase from the maize pathogen Cochliobolus carbonum was cloned using a synthetic, degenerate oligonucleotide based on a tryptic fragment from the purified enzyme. The deduced product of XYL1 has a M(r) of 20,869 and a predicted pI of 9.1, in good agreement with the measured M(r) and pI of the purified enzyme. The XYL1 product has strong amino acid identity to seven endo-beta 1,4-xylanases from six prokaryotes but no obvious similarity to 10 other prokaryotic endoxylanases or a yeast endoxylanase. An internal fragment of the gene was used to create a specific xylanase mutant by transformation-mediated gene disruption via homologous recombination. Total extracellular xylanase activity in the mutant was reduced by 85-94%. When analyzed by cation exchange HPLC, culture filtrates of the mutant and wild type had identical protein profiles, but the mutant lacked the major peak of UV absorption corresponding to the major xylanase activity. Xylanase II activity was also missing in the mutant, but xylanase III activity was still present. The XYL1 mutant grew as well as the wild type on sucrose, on corn cell walls, and on xylan. The pathogenicity of the mutant was indistinguishable from the wild type, indicating that XYL1 is not required for pathogenicity.
Cellulase activity assay on carboxymethylcellulose (CMC) agar plate tests in (A) Meloidogyne incognita second-stage juvenile (J2) total homogenate, (B) Caenorhabditis elegans total homogenate, and (C) M. incognita J2 stylet secretions. For each sample, 6.5 µg of protein was loaded in a 2-µl spot. The same volume was spotted for the (D) secretion negative control. Escherchia coli clones obtained from the J2 cDNA library (E) containing the Mi-eng1 cDNA and (F) not carrying an endoglucanase cDNA, grown on Luria-Bertani (LB) medium, 0.% CMC, supplemented with ampicillin and isopropyl-β-D-thiogalactopyranoside. G, Eggs embedded in their gelatinous matrix directly loaded on the CMC plate. H, Gelatinous matrix devoid of eggs. I, Whole eggs cleaned from gelatinous matrix. 
Southern blot probed at 55°C with the radiolabeled β-1,4endoglucanase 650-bp genomic amplification product from Meloidogyne incognita. Genomic DNA (10 µg each) from (lane 1) M. incognita, (lane 2) M. javanica, (lane 3) M. arenaria, and (lane 4) M. hapla was digested with EcoRI and HindIII prior to separation for Southern analyses. 
Meloidogyne incognita Mi-eng-1 cDNA sequence and predicted amino acid sequence. Predicted secretion signal sequence and amino acids from the catalytic site are indicated in boxes. The linker separating the N-terminal catalytic domain from the C-terminal cellulose-binding domain is underlined. The potential N-glycosylation site is double underlined. Predicted locations of the two intronic sequences identified are indicated by darkened triangles. Primers used for genomic amplification and reverse transcription-polymerase chain reaction are indicated by arrows. The nucleotide sequence is available in the GenBank data base under the accession number AF100549. 
Detection of Mi-eng-1 transcripts in (lane 1) eggs, (lane 2) preparasitic second-stage juveniles (J2), (lane 3) females, (lane 4) males, and (lane 5) uninfected tomato roots by reverse transcriptionpolymerase chain reaction with endoglucanase primers. Equivalent amounts of RNA were used for synthesis of first-strand cDNAs. Amplification of a 324-bp fragment in the catalytic domain visualized on (A) agarose gel and (B) transferred onto Hybond N + membrane and probed with Mi-eng-1 catalytic domain. MW: molecular weight marker. 
Localization of β-1,4-endoglucanase transcripts in the subventral glands of Meloidogyne incognita second-stage juveniles. Fixed nematodes were cut in two to three sections and incubated with (A) antisense or (B) sense riboprobes from Mi-eng-1 catalytic domain. Horizontal bars equal 0.01 mm. 
A beta-1,4-endoglucanase encoding cDNA (EGases, E.C., named Mi-eng-1, was cloned from Meloidogyne incognita second-stage juveniles (J2). The deduced amino acid sequence contains a catalytic domain and a cellulose-binding domain separated by a linker. In M. incognita, the gene is transcribed in the migratory J2, in males, and in the sedentary adult females. In pre-parasitic J2, endoglucanase transcripts are located in the cytoplasm of the subventral esophageal glands. The presence of beta-1,4-endoglucanase transcripts in adult females could be related to the expression of the gene in esophageal glands at this stage. However, cellulase activity within the egg matrix of adult females suggests that the endoglucanase may also be synthesized in the rectal glands and involved in the extrusion of the eggs onto the root surface. The maximum identity of the predicted MI-ENG-1 catalytic domain with the recently cloned cyst nematode beta-1,4-endoglucanases is 52.5%. In contrast to cyst nematodes, M. incognita pre-parasitic J2 were not found to express a beta-1,4-endoglucanase devoid of a cellulose-binding domain.
Phytopathogenic fungi can degrade xylan, an abundant hemicellulose in plant cell walls, by the coordinate action of a group of extracellular enzymes. Among these, endo-beta-1,4-xylanases carry out the initial breakdown by cleaving internal bonds in the polymer backbone. We have isolated and characterized a gene, xyn11A, coding for an endo-beta-1,4-xylanase belonging to family 11 of glycosyl hydrolases. xyn11A was shown to be induced by xylan and repressed by glucose and to be expressed in planta. The disruption of xyn11A caused only a moderate decrease, about 30%, in the level of extracellular endo-beta-1-4-xylanase activity and in the growth rate, with beechwood xylan as the only carbon source. However, deletion of the gene had a more pronounced effect on virulence, delaying the appearance of secondary lesions and reducing the average lesion size by more than 70%. Reintroducing the wild-type gene into the mutant strains reversed this phenotype back to wild type.
Phosphoinositide-specific phospholipase C (PI-PLC) has been shown to be transiently activated when plant cells were treated with elicitors. We thus investigated the activity of PI-PLC when soybean cells were infected with the bacterial pathogen Pseudomonas syringae pv. glycinea, by measuring cellular cytosolic inositol 1,4,5-trisphosphate (IP3) levels. We observed that IP3 content decreased in both compatible and incompatible interactions. In vitro phosphatase activities were similar in both water control and infected cells with slightly lower IP3 degradation observed for infected cells, indicating that the reduced IP3 content in infected cells most likely results from reduced PI-PLC activity. We hypothesize that reduced IP3 content following infection may lead to suppression of various housekeeping activities of the cells, thus diverting the cellular resources either to the synthesis of defense-related compounds against pathogens, and/or to the growth of pathogens.
Mi-1.2, a member of the intracellular, nucleotide-binding site-leucine-rich repeat family of resistance genes, confers resistance in tomato (Lycopersicon esculentum) against both root-feeding nematodes and leaf-feeding aphids. Nematode resistance is effective in all life stages of the plant; in contrast, Mi-mediated aphid resistance is developmentally regulated, and protects mature plants but not seedlings against aphid infestation. To determine if the onset of aphid resistance is regulated by Mi-1.2 transcript abundance, we compared aphid resistance and Mi-1.2 transcript levels in seedlings and flowering plants. Paired bioassays and RNase protection assays revealed that Mi-1.2 is transcribed in the leaves prior to the onset of aphid resistance, and that transcript levels are comparable in seedlings and flowering life stages. Furthermore, constitutive overexpression of Mi-1.2 in transgenic plants did not hasten the onset of aphid resistance in seedlings, or boost the level of resistance observed in flowering plants. These data demonstrate that Mi-1.2 transcript levels do not modulate the degree of aphid resistance in tomato leaves, or control the differences in regulation between aphid and nematode resistance.
The Mi-1.2 gene in tomato (Solanum lycopersicum) is a member of the nucleotide-binding leucine-rich repeat (NBLRR) class of plant resistance genes, and confers resistance against root-knot nematodes (Meloidogyne spp.), the potato aphid (Macrosiphum euphorbiae), and the sweet potato whitefly (Bemisia tabaci). Mi-1.2 mediates a rapid local defensive response at the site of infection, although the signaling and defensive pathways required for resistance are largely unknown. In this study, eggplant (S. melongena) was transformed with Mi-1.2 to determine whether this gene can function in a genetic background other than tomato. Eggplants that carried Mi-1.2 displayed resistance to the root-knot nematode Meloidogyne javanica but were fully susceptible to the potato aphid, whereas a susceptible tomato line transformed with the same transgene was resistant to nematodes and aphids. This study shows that Mi-1.2 can confer nematode resistance in another Solanaceous species. It also indicates that the requirements for Mi-mediated aphid and nematode resistance differ. Potentially, aphid resistance requires additional genes that are not conserved between tomato and eggplant.
Adults and pupae of the B-biotype of Bemisia tabaci a 
L3, pupae, and adults (or empty pupal cases) of the B-and Qbiotype of Bemisia tabaci a 
Averaged daily temperatures recorded in the greenhouse during the two free-choice experiments with the B-biotype carried out under normal (N) and high (H) temperatures, respectively.  
The tomato gene Mi-1.2 confers resistance against root-knot nematodes and some isolates of potato aphid. Resistance to the whitefly Bemisia tabaci previously has been observed in Mi-bearing commercial tomato cultivars, suggesting that Mi, or a closely linked gene, is responsible for the resistance. The response of two biotypes of B. tabaci to tomato carrying the cloned Mi was compared with that of the isogenic untransformed tomato line Moneymaker. Our results indicate that Mi-1.2 is responsible for the resistance in tomato plants to both B- and Q- biotypes. Mi-1.2 is unique among characterized resistance genes in its activity against three very different organisms (root-knot nematodes, aphids, and whiteflies). These pests are among the most important on tomato crops worldwide, making Mi a valuable resource in integrated pest management programs.
Plant resistance (R) proteins mediate race-specific immunity and initiate host defenses that are often accompanied by a localized cell-death response. Most R proteins belong to the nucleotide binding-leucine-rich repeat (NB-LRR) protein family, as they carry a central NB-ARC domain fused to an LRR domain. The coiled-coil (CC) domain at the N terminus of some solanaceous NB-LRR proteins is extended with a solanaceae domain (SD). Tomato Mi-1.2, which confers resistance against nematodes, white flies, psyllids, and aphids, encodes a typical SD-CNL protein. Here, we analyzed the role of the extended N terminus for Mi-1.2 activation. Removal of the first part of the N terminus (Nt1) induced Mi-1.2-mediated cell death that could be suppressed by overexpression of the second half of the N-terminal region. Yet, autoactivating NB-ARC-LRR mutants require in trans coexpression of the N-terminal region to induce cell death, indicating that the N terminus functions both as a negative and as a positive regulator. Based on secondary structure predictions, we could link both activities to three distinct subdomains, a typical CC domain and two novel, structurally-conserved helical subdomains called SD1 and SD2. A negative regulatory function could be assigned to the SD1, whereas SD2 and the CC together function as positive regulators of Mi-1.2-mediated cell death.
Incubation with cucumber phloem exudate in vitro results in a dramatic decrease in the electrophoretic mobility of Hop stunt viroid. UV cross-linking and a combination of size exclusion and ion exchange chromatography indicate that this phenomenon reflects a previously unsuspected ability of phloem protein 2, a dimeric lectin and the most abundant component of phloem exudate, to interact with RNA. In light of its demonstrated ability to move from cell to cell via plasmodesmata as well as long distances in the phloem, our results suggest that phloem protein 2 may facilitate the systemic movement of viroids and, possibly, other RNAs in vivo.
A gene library of the symbiotic 240-kb plasmid of Rhizobium leguminosarum strain 1001 was constructed in pUC18. The clones showing homology with a 6.6-kb fragment containing nodEFDABC from the Sym plasmid pRLlJI were detected by colony hybridization. Additional probes from the symbiotic region of pRLlJI were used to localize the corresponding genes on the map of pRle1001a. The relative positions of nod and nif gene clusters are different than those of pRLlJI. A comparison of the amino acid sequence for NodD from pRle1001a with NodD proteins from other Rhizobium species showed a high degree of sequence conservation at the amino terminus of the protein.
Gas chromatograph with flame ionization detection traces of lipid A constituents obtained after acidic methanolysis and silylation. A, Rm1021, B, EK257, C, EK273; 1, 4, and 5 = methyl-trimethylsilyl- glucosamine, 2 = nC16 fatty acid methyl ester (FAME), 3 = 3-OH-C14 FAME, 6 = nC18 FAME, 7 = 3-OH-C16 FAME, 8 = 3-OH-C18 FAME, and 9 = 27-OH-C28 FAME. 
Nitrogen fixation efficiency of S. meliloti strains on different host plants in the presence or absence of the functional typA gene
Medicago truncatula lines inoculated with the wild-type (left), typA mutant (middle), and typA mutants carrying the complementing plasmid (right) A, DZA315.16, B, F83005.5, and C, Jemalong. 
lacZ staining of nodules inoculated by pXLGD4-containing bacteria. A and B, Jemalong 6 days postinoculation (dpi), C and D, Jemalong 30 dpi, E and F, F83005.5 6 dpi, G and H, F83005.5 30 dpi; A, C, E, and G, inoculated with Rm1021 and B, D, F, and H, inoculated with the typA mutant. Bar = 100 μm (A, B, D, E, and F) and 200 μm (C, G, and H). 
Localization of typA expression in Medicago truncatula DZA315.16 nodules A, 6 days postinoculation (dpi), bar = 100 µm and B, 18 dpi. II = Invasion zone, III = nitrogen fixation zone, and IV = senescent zone. Bar = 200 µm.
In this article, we describe the typA gene of Sinorhizobium meliloti, the orthologue of typA/bipA genes found in a wide range of bacteria. We found that typA was required for survival of S. meliloti under certain stress conditions, such as growth at low temperature or low pH and in the presence of sodium dodecyl sulfate (SDS). The cold-sensitive phenotype of both Escherichia coli bipA and S. meliloti typA mutants were cross-complemented, indicating that the two genes are functionally equivalent. typA was indispensable for symbiosis on Medicago truncatula Jemalong and F83005.5 and contributes to the full efficiency of symbiosis on other host plant lines such as DZA315.16 or several cultivars of M. sativa. Hence, the symbiotic requirement for typA is host dependent. Interestingly, the symbiotic defect was different on Jemalong and F83005.5 plants, thus indicating that typA is required at a different stage of the symbiotic interaction.
In the Rhizobium-legume symbiosis, compatible partners recognize each other through an exchange of signals. Plant inducers act together with bacterial transcriptional activators, the NodD proteins, to regulate the expression of bacterial biosynthetic nodulation (nod) genes. These genes direct the synthesis of a lipochito-oligosaccharide signal called Nod factor (NF). NFs elicit an early host response, root hair calcium spiking, that is initiated in root hair cells within 15 min of NF or live Rhizobium inoculation. We used calcium spiking as an assay to compare two closely related strains of Sinorhizobium meliloti, Rm1021 and Rm2011, derived from the same field isolate. We found that the two strains show a kinetic difference in the calcium spiking assay: Rm1021 elicits calcium spiking in host root hairs as rapidly as purified NF, whereas Rm2011 shows a significant delay. This difference can be overcome by raising expression levels of either the NodD transcriptional activators or GroEL, a molecular chaperone that affects expression of the biosynthetic nod genes. We further demonstrate that the delay in triggering calcium spiking exhibited by Rm2011 is correlated with a reduced amount of nod gene expression compared with Rm1021. Therefore, calcium spiking is a useful tool in detecting subtle differences in bacterial gene expression that affect the early stages of the Rhizobium-legume symbiosis.
Bacterial two-component regulatory systems (TCS) are common components of complex regulatory networks and cascades. In Sinorhizobium meliloti, the TCS ExoS/ChvI controls exopolysaccharide succinoglycan production and flagellum biosynthesis. Although this system plays a crucial role in establishing the symbiosis between S. meliloti and its host plant, it is not well characterized. Attempts to generate complete loss-of-function mutations in either exoS or chvI in S. meliloti have been unsuccessful; thus, it was previously suggested that exoS or chvI are essential genes for bacterial cell growth. We constructed a chvI mutant by completely deleting the open reading frame encoding this gene. The mutant strain failed to grow on complex medium, exhibited lower tolerance to acidic condition, produced significantly less poly-3-hydroxybutyrate than the wild type, was hypermotile, and exhibited an altered lipopolysaccharide profile. In addition, this mutant was defective in symbiosis with Medicago truncatula and M. sativa (alfalfa), although it induced root hair deformation as efficiently as the wild type. Together, our results demonstrate that ChvI is intimately involved in regulatory networks involving the cell envelope and metabolism; however, its precise role within the regulatory network remains to be determined.
Rhizosphere growth limitations imposed on Rhizobium meliloti by availability of biotin, thiamine, and riboflavin were overcome by adding nanomolar amounts of these vitamins. Studies done with R. meliloti 1021 showed that both synthesis and uptake of biotin promote colonization of alfalfa roots. Two lines of evidence indicated that plant-derived biotin normally promotes root colonization: (i) adding avidin significantly (P < or = 0.01) reduced rhizosphere growth of R meliloti 1021, and (ii) growth of Tn5-induced biotin auxotrophs still increased 10-fold in the rhizosphere. Synthesis, however, is the more important source of biotin for R. meliloti 1021 because in root colonization tests biotin auxotrophs competed very poorly with the parent strain. Mutations conferring biotin auxotrophy were closely linked on a single restriction fragment, and one was complemented with the Escherichia coli bio operon. Initial nucleotide sequencing and DNA-DNA hybridization tests showed the biotin synthesis genes in R. meliloti are quite different from those in E. coli.
The cyclic beta-1,2-glucans of Rhizobium may function during legume nodulation. These molecules may become highly substituted with phosphoglycerol moieties from the head group of phosphatidylglycerol; diglyceride is a by-product of this reaction (K. J. Miller, R. S. Gore, and A. J. Benesi, J. Bacteriol. 170:4569-4575, 1988). We recently reported that R. meliloti 1021 produces a diacylglycerol kinase (EC activity that shares several properties with the diacylglycerol kinase enzyme of Escherichia coli (W. P. Hunt, R. S. Gore, K. J. Miller, Appl. Environ. Microbiol. 57:3645-3647, 1991). A primary function of this rhizobial enzyme is to recycle diglyceride generated during cyclic beta-1,2-glucan biosynthesis. In the present study, we report the cloning and initial characterization of a single-copy gene from R. meliloti 1021 that encodes a diacylglycerol kinase homolog; this homolog can complement a diacylglycerol kinase deficient strain of E. coli. The sequence of the rhizobial diacylglycerol kinase gene was predicted to encode a protein of 137 amino acids; this protein shares 32% identity with the E. coli enzyme. Analysis of hydropathy and the potential to form specific secondary structures indicated a common overall structure for the two enzymes. Because diglyceride metabolism and cyclic beta-1,2-glucan biosynthesis are metabolically linked, future studies with diacylglycerol kinase mutants of R. meliloti 1021 should further elucidate the roles of the cyclic beta-1,2-glucans in the Rhizobium-legume symbiosis.
Number of genes found differentially expressed in A, cells grown in tryptone yeast versus Vincent minimal medium (VMM) media, B, bacteroids versus cells cultivated in oxic VMM, and C, cells grown under microoxic versus oxic conditions. Black boxes = common genes found with both micro- and macroarrays and lined boxes = differentially expressed genes identified solely in micro- or macroarrays but supported by one of the following criteria: i) genes found in a putative operon with or in 
Replicon assignment of induced and repressed genes under A, tryptone yeast cultures versus Vincent minimal medium (VMM) cultures, B, bacteroids versus VMM-cultured cells, and C, microoxic versus oxic conditions. Lined boxes = chromosomal genes, black boxes = genes on pSymA, and white boxes = genes on pSymB. 
Position of genes induced in microoxic conditions and in bacteroids on pSymA. Genes induced under microoxic conditions are indicated by red lines. Genes induced in bacteroids are indicated by green lines. Genes induced under both microoxic and symbiotic conditions are indicated by black lines.
Primers used for real-time reverse-transcriptase polymerase chain reaction
Transcript levels of genes detected as differential by arrays and real-time reverse-transcriptase polymerase chain reaction (RT-PCR) experiments in cells cultured in tryptone yeast rich and in Vincent minimal medium
Sinorhizobium meliloti is an alpha-proteobacterium that alternates between a free-living phase in bulk soil or in the rhizosphere of plants and a symbiotic phase within the host plant cells, where the bacteria ultimately differentiate into nitrogen-fixing organelle-like cells, called bacteroids. As a step toward understanding the physiology of S. meliloti in its free-living and symbiotic forms and the transition between the two, gene expression profiles were determined under two sets of biological conditions: growth under oxic versus microoxic conditions, and in free-living versus symbiotic state. Data acquisition was based on both macro- and microarrays. Transcriptome profiles highlighted a profound modification of gene expression during bacteroid differentiation, with 16% of genes being altered. The data are consistent with an overall slow down of bacteroid metabolism during adaptation to symbiotic life and acquisition of nitrogen fixation capability. A large number of genes of unknown function, including potential regulators, that may play a role in symbiosis were identified. Transcriptome profiling in response to oxygen limitation indicated that up to 5% of the genes were oxygen regulated. However, the microoxic and bacteroid transcriptomes only partially overlap, implying that oxygen contributes to a limited extent to the control of symbiotic gene expression.
Isolates of Magnaporthe oryzae (the causal agent of rice blast disease) can infect a range of grass species, including barley. We report that barley Hordeum vulgare cv. Baronesse and an experimental line, BCD47, show a range of resistance reactions to infection with two rice blast isolates. The complete resistance of Baronesse to the isolate Ken 54-20 is controlled by a single dominant gene, designated RMo1. RMo1 mapped to the same linkage map position on chromosome 1H as the powdery mildew resistance locus Mla and an expressed sequence tag (k04320) that corresponds to the barley gene 711N16.16. A resistance quantitative trait locus (QTL), at which Baronesse contributed the resistance allele, to the isolate Ken 53-33 also mapped at the same position as RMo1. Synteny analysis revealed that a corresponding region on rice chromosome 5 includes the bacterial blight resistance gene xa5. These results indicate that a defined region on the short arm of barley chromosome 1H, including RMo1 and Mla, harbors genes conferring qualitative and quantitative resistance to multiple pathogens. The partial resistance of BCD47 to Ken53-33 is determined by alleles at three QTL, two of which coincide with the linkage map positions of the mildew resistance genes mlo and Mlf.
Six-week-old spring wheat Triticum aestivum L. cv. Trenton inoculated with Klebsiella pneumoniae strain 342 (Kp342) and nifH mutant of Kp342 ( nifH ) grown in labeled (10 mg of 15 NH 4 NO 3 per kilogram of soil mix) A, sand and B, perlite. The plants in the three pots on the left were inoculated with the nifH mutant of Kp342 while the plants in the three pots on the right were inoculated with Kp342. C, Chlorophyll readings of 6-week-old spring wheat Triticum aestivum L. cv. Trenton were obtained from Minolta SPAD 502, where the ratio of transmittance from two wavelengths (650/940 nm) creates an arbitrary unit related to chlorophyll content. Plants were inoculated with Kp342, the nifH mutant, or uninoculated (Uninoc.). Treated plants were grown in labeled (10 mg of 15 NH 4 NO 3 per kilogram of soil mix) sand-perlite (open column) or sand-vermiculite (closed column). The columns represent the mean SPAD readings. The bars represent the standard error. Least significant difference statistical analysis was calculated to determine difference between treatments. These differences are represented by letters inside the columns. 
Comparison of three wheat cultivars for their ability to enhance growth in the greenhouse and relieve nitrogen-deficiency symptoms upon inoculation with Klebsiella pneumoniae 342 (Kp342) a
Triticum aestivum L. cv. Trenton plants were inoculated with Klebsiella pneumoniae strain 342 (Kp342) and compared with uninoculated plants or inoculated with a nifH mutant. Dry roots (open columns) and shoots (closed columns) from plants grown in labeled (10 mg of 15 NH 4 NO 3 per kilogram of soil mix) A, C, E, and G, sand-perlite and B, D, F, and H, sand-vermiculite were used to estimate A and B, dry weights, C and D, total N per plant, and total N concentration in E and F, shoots and G and H, roots in dried tissue, 6 weeks post inoculation. The columns represent the mean of dry weight for plants grown in A, sand-perlite and B, sand-vermiculite, and total nitrogen per plant grown in B, sand-perlite and C, sand-vermiculite. The columns also represent the mean total N concentration per gram of E, dried shoot and G, root for plants grown in F, sand-perlite and H, sand-vermiculite. The bars represent the standard error. Least significant difference (LSD) statistical analysis was calculated to determine difference between treatments. Letters inside the columns represents the LSD calculations (roman for roots and italics for shoots). 
A, Percent 15 N content in 6-week-old Triticum aestivum L. cv. Trenton grown in labeled ( 15 NH 4 NO 3 , 11.7 atom percent excess, 10 mg per kilogram of soil mix) sand-perlite ( A, solid open and B, closed columns) and sand-vermiculite ( A, dotted open and B, closed columns). The percent 15 N was analyzed 
A through D, Comparison of A and C, green fluorescent protein (GFP)-labeled (green) Klebsiella pneumoniae 342 (Kp342) wild-type and B and D, GFP-labeled (green) Kp342 nifH mutant of spring wheat Triticum aestivum L . cv. Trenton root colonization. A and B, Cross sections of spring wheat roots were examined as well as C and D, lateral root emergence Bars (50 μm). E, Immunolocalization of NifH produced by GFP-labeled Kp342 in root cross section. Cells are seen in yellow as the fluorophores of NifH (red) and GFP-labeled Kp342 are colocalized (yellow) Bars (50 um). F, Number of CFU recovered from the interior of roots T. aestivum L . cv. Trenton. Plants were inoculated with K. pneumoniae strain 342 (closed columns) and nifH mutant of Kp342 (open column) at 10 2 and 10 4 CFU/plant inoculum level. The columns represent the means of each treatment. Each treatment consists of four replicates and each replicate consists of four plants. The bars represent the standard errors about the mean; gfw, gram (fresh weight). 
In this report, all of the criteria necessary for the demonstration of nitrogen fixation in wheat (Triticum aestivum L.), the world's most important crop, are shown upon inoculation with a nitrogen-fixing bacterium, Klebsiella pneumoniae 342 (Kp342). Kp342 relieved nitrogen (N) deficiency symptoms and increased total N and N concentration in the plant. Nitrogen fixation was confirmed by 15N isotope dilution in the plant tissue and in a plant product, chlorophyll. All of these observations were in contrast to uninoculated plants, plants inoculated with a nitrogen-fixing mutant of Kp342, and plants inoculated with dead Kp342 cells. Nitrogenase reductase was produced by Kp342 in the intercellular space of the root cortex. Wild-type Kp342 and the nifH mutant colonized the interior of wheat roots in equal numbers on a fresh weight basis. The nitrogen fixation phenotype described here was specific to cv. Trenton. Inoculation of cvs. Russ or Stoa with Kp342 resulted in no relief of nitrogen deficiency symptoms.
Many gram-negative bacteria regulate expression of specialized gene sets in response to population density. This regulatory mechanism, called autoinduction or quorum-sensing, is based on the production by the bacteria of a small, diffusible signal molecule called the autoinducer. In the most well-studied systems the autoinducers are N-acylated derivatives of L-homoserine lactone (acyl-HSL). Signal specificity is conferred by the length, and the nature of the substitution at C-3, of the acyl side-chain. We evaluated four acyl-HSL bioreporters, based on tra of Agrobacterium tumefaciens, lux of Vibrio fischeri, las of Pseudomonas aeruginosa, and pigment production by Chromobacterium violaceum, for their ability to detect sets of 3-oxo acyl-HSLs, 3-hydroxy acyl-HSLs, and alkanoyl-HSLs with chain lengths ranging from C4 to C12. The traG::lacZ fusion reporter from the A. tumefaciens Ti plasmid was the single most sensitive and versatile detector of the four. Using this reporter, we screened 106 isolates representing seven genera of bacteria that associate with plants. Most of the Agrobacterium, Rhizobium, and Pantoea isolates and about half of the Erwinia and Pseudomonas isolates gave positive reactions. Only a few isolates of Xanthomonas produced a detectable signal. We characterized the acyl-HSLs produced by a subset of the isolates by thin-layer chromatography. Among the pseudomonads and erwinias, most produced a single dominant activity chromatographing with the properties of N-(3-oxo-hexanoyl)-L-HSL. However, a few of the erwinias, and the P. fluorescens and Ralstonia solanacearum isolates, produced quite different signals, including 3-hydroxy forms, as well as active compounds that chromatographed with properties unlike any of our standards. The few positive xanthomonas, and almost all of the agrobacteria, produced small amounts of a compound with the chromatographic properties of N-(3-oxo-octanoyl)-L-HSL. Members of the genus Rhizobium showed the greatest diversity, with some producing as few as one and others producing as many as seven detectable signals. Several isolates produced extremely nonpolar compounds indicative of very long acyl side-chains. Production of these compounds suggests that quorum-sensing is common as a gene regulatory mechanism among gram-negative plant-associated bacteria.
Exopolysaccharide (EPS) and lipopolysaccharide (LPS) from Bradyrhizobium japonicum are important for infection and nodulation of soybean (Glycine max), although their roles are not completely understood. To better understand this, we constructed mutants in B. japonicum USDA 110 impaired in galactose or galacturonic acid incorporation into the EPS without affecting the LPS. The derivative LP 3010 had a deletion of lspL-ugdH and produced EPS without galacturonic acid whereas LP 3013, with an insertion in exoB, produced EPS without galactose. In addition, the strain LP 3017, with both mutations, had EPS devoid of both galactosides. The missing galactosides were not replaced by other sugars. The defects in EPS had different consequences. LP 3010 formed biofilms and nodulated but was defective in competitiveness for nodulation; and, inside nodules, the peribacteroid membranes tended to fuse, leading to the merging of symbiosomes. Meanwhile, LP 3013 and LP 3017 were unable to form biofilms and produced empty pseudonodules but exoB suppressor mutants were obtained when LP 3013 plant inoculation was supplemented with wild-type EPS. Similar phenotypes were observed with all these mutants in G. soja. Therefore, the lack of each galactoside in the EPS has a different functional effect on the B. japonicum-soybean symbiosis.
Analysis of the expression levels of 11 Arabidopsis thaliana ubiquitin-ligase genes after chitooctaose treatment. Quantitative polymerase chain reactions were performed on cDNA isolated from plants treated with chitooctaose (left to right) 15 (blue), 30 (purple), 60 (yellow), 90 (blue light), and 120 (dark purple) min. The gene designations are shown on the x axis, while the y axis presents the ratio of the expression levels (chitooctaose treatment/mock).  
Comparison of the expression levels of Arabidopsis thaliana ubiquitin-ligase and transcription factor genes after chitotetraose and chitooctaose treatment. Quantitative polymerase chain reactions were performed on cDNA isolated from plants treated 30 min with chitotetraose (gray) or chitooctaose (white) at a final concentration of 1 μM. The gene designations are shown on the x axis, while the y axis presents the ratio of the expression levels (chitin treatment/mock).  
Chitin, found in the cell walls of true fungi and the exoskeleton of insects and nematodes, is a well-established elicitor of plant defense responses. In this study, we analyzed the expression patterns of Arabidopsis thaliana transcription factor (TF) and ubiquitin-ligase genes in response to purified chitooctaose at different treatment times (15, 30, 60, 90, and 120 min after treatment), using both quantitative polymerase chain reaction and the Affymetrix Arabidopsis whole-genome array. A total of 118 TF genes and 30 ubiquitin-ligase genes were responsive to the chitin treatment. Among these genes, members from the following four TF families were overrepresented: APETALA2/ethylene-reponsive element binding proteins (27), C2H2 zinc finger proteins (14), MYB domain-containing proteins (11), and WRKY domain transcription factors (14). Transcript variants from a few of these genes were found to respond differentially to chitin, suggesting transcript-specific regulation of these TF genes.
G protein signaling is commonly involved in regulating growth and differentiation of eukaryotic cells. We previously identified MAGB, encoding a Galpha subunit, from Magnaporthe grisea, and disruption of MAGB led to defects in a number of cellular responses, including appressorium formation, conidiation, sexual development, mycelial growth, and surface sensing. In this study, site-directed mutagenesis was used to further dissect the pleiotropic effects controlled by MAGB. Conversion of glycine 42 to arginine was predicted to abolish GTPase activity, which in turn would constitutively activate G protein signaling in magB(G42R). This dominant mutation caused autolysis of aged colonies, misscheduled melanization, reduction in both sexual and asexual reproduction, and reduced virulence. Furthermore, magB(G42R) mutants were able to produce appressoria on both hydrophobic and hydrophilic surfaces, although development on the hydrophilic surface was delayed. A second dominant mutation, magB(G203R) (glycine 203 converted to arginine), was expected to block dissociation of the Gbetagamma from the Galpha subunit, thus producing a constitutively inactive G protein complex. This mutation did not cause drastic phenotypic changes in the wild-type genetic background, other than increased sensitivity to repression of conidiation by osmotic stress. However, magB(G203R) is able to complement phenotypic defects in magB mutants. Comparative analyses of the phenotypical effects of different magB mutations are consistent with the involvement of the Gbetagamma subunit in the signaling pathways regulating cellular development in M. grisea.
The Tobacco mosaic virus (TMV) 126-kDa protein is a suppressor of RNA silencing previously shown to delay the silencing of transgenes in Nicotiana tabacum and N. benthamiana. Here, we demonstrate that expression of a 126-kDa protein-green fluorescent protein (GFP) fusion (126-GFP) in N. tabacum increases susceptibility to a broad assortment of viruses, including Alfalfa mosaic virus, Brome mosaic virus, Tobacco rattle virus (TRV), and Potato virus X. Given its ability to enhance TRV infection in tobacco, we tested the effect of 126-GFP expression on TRV-mediated virus-induced gene silencing (VIGS) and demonstrate that this protein can enhance silencing phenotypes. To explain these results, we examined the poorly understood effect of suppressor dosage on the VIGS response and demonstrated that enhanced VIGS corresponds to the presence of low levels of suppressor protein. A mutant version of the 126-kDa protein, inhibited in its ability to suppress silencing, had a minimal effect on VIGS, suggesting that the suppressor activity of the 126-kDa protein is indeed responsible for the observed dosage effects. These findings illustrate the sensitivity of host plants to relatively small changes in suppressor dosage and have implications for those interested in enhancing silencing phenotypes in tobacco and other species through VIGS.
Systemic symptoms induced on Nicotiana tabacum cv. Xanthi by Tobacco mosaic virus (TMV) are modulated by one or both amino-coterminal viral 126- and 183-kDa proteins: proteins involved in virus replication and cell-to-cell movement. Here we compare the systemic accumulation and gene silencing characteristics of TMV strains and mutants that express altered 126- and 183-kDa proteins and induce varying intensities of systemic symptoms on N. tabacum. Through grafting experiments, it was determined that M(IC)1,3, a mutant of the masked strain of TMV that accumulated locally and induced no systemic symptoms, moved through vascular tissue but failed to accumulate to high levels in systemic leaves. The lack of M(IC)1,3 accumulation in systemic leaves was correlated with RNA silencing activity in this tissue through the appearance of virus-specific, approximately 25-nucleotide RNAs and the loss of fluorescence from leaves of transgenic plants expressing the 126-kDa protein fused with green fluorescent protein (GFP). The ability of TMV strains and mutants altered in the 126-kDa protein open reading frame to cause systemic symptoms was positively correlated with their ability to transiently extend expression of the 126-kDa protein:GFP fusion and transiently suppress the silencing of free GFP in transgenic N. tabacum and transgenic N. benthamiana, respectively. Suppression of GFP silencing in N. benthamiana occurred only where virus accumulated to high levels. Using agroinfiltration assays, it was determined that the 126-kDa protein alone could delay GFP silencing. Based on these results and the known synergies between TMV and other viruses, the mechanism of suppression by the 126-kDa protein is compared with those utilized by other originally characterized suppressors of RNA silencing.
Pseudomonas syringae pv. tomato DC3000 inoculation strongly increases NaPR-1 and NaThionin transcripts in wild-type (WT) Nicotiana attenuata. Quantitative real-time reverse-transcriptase polymerase chain reaction was used to analyze NaPR-1 and NaThionin transcript accumulation in WT N. attenuata plants in response to inoculation with P. syringae pv. tomato DC3000 (1 × 10 5 cells/ml) or water. Values are mean (± standard error) of NaPR-1 and NaThionin transcripts from three replicate plants per treatment normalized to the transcript abundance of actin, which is unregulated under these conditions. Asterisks indicate significant differences in NaPR-1 and NaThionin transcript accumulation between P. syringae pv. tomato DC3000-inoculated and waterinoculated WT plants at the designated time, P < 0.001 (***) (n = 3). 
Increases in pathogenesis-related (PR) transcripts are commonly interpreted as evidence of plants' resistance responses to pathogens; however, few studies have examined whether increases in PR proteins protect plants growing under natural conditions. Pseudomonas syringae pv. tomato DC3,000, which is virulent and causes disease in Arabidopsis, is also pathogenic to the native tobacco Nicotiana attenuata. N. attenuata responds to P. syringae pv. tomato DC3,000's challenges with increases in salicylic acid and transcripts of at least two PR genes, PR-1 and PR13/Thionin. To determine if either of these PR proteins functions in bacterial resistance, we independently silenced both genes by RNAi and found that only PR-13/Thionin mediates resistance to P. syringae pv. tomato DC3,000 in glasshouse experiments. When NaPR-1- and NaThionin-silenced plants were transplanted into the plant's native habitat in the Great Basin Desert of Utah, opportunistic Pseudomonas spp. performed better on NaThionin-silenced plants compared with NaPR-1-silenced and wild-type (WT) plants, and accounted for increased plant mortality. The native herbivore community of N. attenuata attacked both NaPR-1- and PR-13/NaThionin-silenced plants to the same degree as it did in WT plants, indicating that neither PR protein provides resistance to herbivores. Although PR-1 is generally considered a marker gene of disease resistance, we found no evidence that it has an antimicrobial function. In contrast, PR-13/NaThionin is clearly an ecologically relevant defense protein involved in resisting pathogens in N. attenuata.
Oxylipins recently have been implicated as signaling molecules for cross-kingdom communication in plant-pathogen interactions. Linoleic acid and its two plant lipoxygenase (LOX) oxylipin products 9- and 13-hydroperoxy fatty acids (9S- and 13S-HPODE) have been shown to have a significant effect on differentiation processes in the mycotoxigenic seed pathogens Aspergillus spp. Whereas both fatty acids promote sporulation, 9S-HPODE stimulates and 13S-HPODE inhibits mycotoxin production. Additionally, Aspergillus flavus infection of seed promotes linoleate 9-LOX expression and 9S-HPODE accumulation. Here, we describe the characterization of two peanut seed lipoxygenase alleles (PnLOX2 and PnLOX3) highly expressed in mature seed. PnLOX2 and PnLOX3 both are 13S-HPODE producers (linoleate 13-LOX) and, in contrast to previously characterized 9-LOX or mixed function LOX genes, are repressed between 5-fold and 250-fold over the course of A. flavus infection. The results of these studies suggest that 9S-HPODE and 13S-HPODE molecules act as putative susceptibility and resistance factors respectively, in Aspergillus seed-aflatoxin interactions.
Microorganisms and plasmids 
Biocontrol of Pseudomonas chlororaphis PCL1391 and its root colonization-impaired derivatives. Strains PCL1391 (wild type), PCL1126 (sss), PCL1128 (phe), and PCL1131 (motility impaired) were 
The phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicislycopersici. To test whether root colonization is required for biocontrol, mutants impaired in the known colonization traits motility, prototrophy for amino acids, or production of the site-specific recombinase, Sss/XerC were tested for their root tip colonization and biocontrol abilities. Upon tomato seedling inoculation, colonization mutants of strain PCL1391 were impaired in root tip colonization in a gnotobiotic sand system and in potting soil. In addition, all mutants were impaired in their ability to control tomato foot and root rot, despite the fact that they produce wild-type levels of phenazine-1-carboxamide, the antifungal metabolite previously shown to be required for biocontrol. These results show, for what we believe to be the first time, that root colonization plays a crucial role in biocontrol, presumably by providing a delivery system for antifungal metabolites. The ability to colonize and produce phenazine-1-carboxamide is essential for control of F. oxysporum f. sp. radicis-lycopersici. Furthermore, there is a notable overlap of traits identified as being important for colonization of the rhizosphere and animal tissues.
The pathogenicity-associated plasmid (pPATH) of Erwinia herbicola pv. gypsophilae was previously shown to be exclusively present in pathogenic strains and to contain a gene cluster encoding phytohormone biosynthesis. Sequence analysis of the DNA region located downstream from the cytokinin biosynthetic gene (etz) revealed homology to insertion sequences (IS) of the IS6 family. Southern blot analysis performed on plasmid DNA of E. herbicola pv. gypsophilae revealed the presence of six copies of this insertion-like element, which was designated as IS1327. Only pathogenic strains contained IS1327 and restriction fragment length polymorphism was observed among gypsophilae and beet pathovars of E. herbicola. Nonpathogenic deletion derivatives of pPATH contained fewer copies of IS1327, suggesting its presence in the deleted region. One copy of IS1327 (IS1327-R) was located 2.8 kb downstream from the IS element adjacent to the etz (IS1327-L) in a direct repeat.
Model for the modulation of host immunity in the ecto-and endomycorrhizal symbioses. A, Root exudates recruit symbiotic mycorrhizal fungi and prime them for the interaction. Host plants initially recognize ectomycorrhizal (EMF) and arbuscular mycorrhizal (AMF) fungi as potential invaders; patternrecognition receptors (PRR) in the host perceive microbe-associated molecular patterns (MAMPs, yellow-colored shapes) and a signaling cascade is initiated that results in MAMP-triggered immunity (MTI). B, The EMF Laccaria bicolor secretes proteins similar to the haustoria-expressed proteins of pathogenic basidiomycetes (SiHEP) and several cysteine-rich mycorrhiza-induced small secreted proteins (MiSSP) that may function as extra-or intracellular effectors to suppress host immune responses. The MiSSP7 effector is perceived by the plant cell through lipid-raft-mediated endocytosis and translocated into the nucleus, where it promotes auxin-related gene expression. It remains to be demonstrated whether upregulation of auxin signaling has a negative impact on plant defenses, as demonstrated for pathogenic interactions. C, In the AM symbiosis, the establishment of the symbiotic program in plant cells, which is activated upon perception of the mycorrhizal Myc factors, counteracts MTI with mechanisms yet to be defined. Molecules (brown-colored shapes) that are secreted in the apoplastic or periarbuscular space (PAS) may act as either apoplastic or cytoplasmic effectors to suppress the MTI response or promote the symbiotic program. The AMF Glomus intraradices secretes the SP7 effector which is translocated into the plant cytosol; a nuclear localization signal (NLS) targets SP7 to the nucleus, where it interacts with the defense-related transcription factor ERF19 to block the ERF19-mediated transcriptional program. 
In nature, plants abundantly form beneficial associations with soilborne microbes that are important for plant survival and, as such, affect plant biodiversity and ecosystem functioning. Classical examples of symbiotic microbes are mycorrhizal fungi that aid in the uptake of water and minerals, and Rhizobium bacteria that fix atmospheric nitrogen for the plant. Several other types of beneficial soilborne microbes, such as plant-growth-promoting rhizobacteria and fungi with biological control activity, can stimulate plant growth by directly suppressing deleterious soilborne pathogens or by priming aboveground plant parts for enhanced defense against foliar pathogens or insect herbivores. The establishment of beneficial associations requires mutual recognition and substantial coordination of plant and microbial responses. A growing body of evidence suggests that beneficial microbes are initially recognized as potential invaders, after which an immune response is triggered, whereas, at later stages of the interaction, mutualists are able to short-circuit plant defense responses to enable successful colonization of host roots. Here, we review our current understanding of how symbiotic and nonsymbiotic beneficial soil microbes modulate the plant immune system and discuss the role of local and systemic defense responses in establishing the delicate balance between the two partners.
Endophytic Pseudomonas poae strain RE*1-1-14 was originally isolated from internal root tissue of sugar beet plants and shown to suppress growth of the fungal pathogen Rhizoctonia solani both in vitro and in the field. To identify genes involved in its biocontrol activity, RE*1-1-14 random mutagenesis and sequencing led to the identification of a nonribosomal peptide synthetase (NRPS) gene cluster predicted to encode a lipopeptide (LP) with a 10-amino acid peptide moiety. The two unlinked gene clusters consisted of three NRPS genes, designated poaA (cluster 1), and poaB and poaC (cluster 2), spanning approximately 33.7 kb. In silico analysis followed by chemical analyses revealed that the encoded LP designated poaeamide, is a structurally new member of the orfamide family. Poaeamide inhibited mycelial growth of R. solani and different oomycetes including Phytophthora capsici, Ph. infestans, and Pythium ultimum. The novel LP was shown to be essential for swarming motility of strain RE*1-1-14 and had an impact on root colonization of sugar beet seedlings The poaeamide-deficient mutant colonized the rhizosphere and upper plant cortex at higher densities and with more scattered colonization patterns than the wildtype. Collectively these results indicate that P. poae RE*1-1-14 produces a structurally new LP that is relevant for its antagonistic activity against soil-borne plant pathogens and for colonization of sugar beet roots.
14-3-3 proteins define a eukaryotic-specific protein family with a general role in signal transduction. Primarily, 14-3-3 proteins act as phospho-sensors, binding phosphorylated client proteins and modulating their functions. Since phosphorylation regulates a plethora of different physiological responses in plants, 14-3-3 proteins play roles in multiple signalling pathways, including those controlling metabolism, hormone signalling, cell division, and responses to abiotic and biotic stimuli. Increasing evidence supports a prominent role of 14-3-3 proteins in regulating plant immunity against pathogens at various levels. In this review, potential links between 14-3-3 function and the regulation of plant-pathogen interactions are discussed, with a special focus on the regulation of 14-3-3s in response to pathogen perception, interactions between 14-3-3s and defence-related proteins, and 14-3-3s as targets of pathogen effectors.
A chimeric vector was constructed to express cucumber mosaic virus (CMV) satellite (Sat) RNA and coat protein (CP). Transgenic lines of tobacco cultivar G-140 expressing CP and Sat-RNA were obtained; these lines had high resistance to CMV. Fifty to 70% of the transgenic plants were symptomless 90 days after inoculation with 25-50 micrograms/ml of CMV. Resistance was about twice that conferred by the Sat-RNA or the CP gene alone in transformed plants.
Expression of the chromosomal foxR9::lacZ fusion in Erwininia amylovora cells grown in (A) Luria-Bertani (LB) medium and (D) after inoculation onto apple seedlings. A, LB supplemented with 50 µM ED- DHA or 50 µM FeCl 3 as indicated, to provide low and rich iron conditions, respectively. Experiment was performed in triplicate; standard deviations are shown. D, β-Galactosidase activity assayed in infected leaves, divided in three batches of five leaves, for 5 days after inoculation . Concurrent enumeration of bacterial populations was determined after plating on L agar medium in the presence and absence of appropriate antibiotics indicating plasmid stability. Correspondant optical densities at 600 nm were assessed as reported in B and C. Two independent assays, (1) and (2), are shown. Control experiments correspond to plant tissues inoculated with the wild-type strain.  
In planta immunofluorescence detection of Erwininia amylovora cells harboring the fusion foxR9::lacZ and pAL2 with a rabbit antilipopolysaccharide (LPS) polyclonal serum and a fluorescein isothiocyanate (FITC) coupled anti-rabbit IgG conjugate. A, General view of a transverse section of a leaf. B, Detail of bacteria in intercellular spaces of the spongy parenchyma (sp) of the lamina. Bacteria (b) are in contact with the plant cell walls. Inset: high magnification showing labeling at the bacterial cell surface. C, Detail of the midrib region with invasion of bacteria in tracheids (x) of the vein. A large, distorted xylem element (dx) is filled with bacteria. l, lamina; lc, lysigenous cavity of the cortical parenchyma; mr, midrib; pc, cortical parenchyma; sp, spongy parenchyma ; Bar = (A) 100 and (B and C) 20 µm.  
In planta expression of the foxR9::lacZ fusion visualized by X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) staining. Sections poststained for 15 s with 0.1% aqueous fuchsin. A, General view showing expression in lysigenous cavities (lc) of cortical parenchyma (pc) and in distorted xylem element (dx) of midrib vein. B, Detail of a lysigenous cavity of the cortical parenchyma invaded with bacteria expressing different levels of βgalactosidase activity. It is surrounded by a thick cell wall (tw). C, Detail of the midrib vein showing some reactive bacteria (rb) expressing βgalactosidase in a distorted xylem element (dx). Conversely, bacteria present in a neighboring xylem element (x) are unreactive (ur). bst, broken secondary thickenings of xylem wall; l, lamina; st, secondary thickenings. Bar = (A) 100 and (B and C) 20 µm.  
Mutants of Erwinia amylovora CFBP 1430 lacking a functional high-affinity iron transport system mediated by desferrioxamine are impaired in their ability to initiate fire blight symptoms (A. Dellagi, M.-N. Brisset, J.-P. Paulin, and D. Expert. Mol. Plant-Microbe Interact. 11:734-742, 1998). In this study, a chromosomal transcriptional lacZ fusion was used to analyze the expression in planta of the E. amylovora ferrioxamine receptor gene foxR. LacZ activity produced by the strain harboring the fusion was highly induced in iron-restricted conditions and in inoculated apple leaf tissues. Microscopic observation revealed differential expression of this gene in relation to the localization and density of bacterial cells within the diseased tissue. Thus, the ability of bacterial cells to express their iron transport system in accordance with environmental conditions is likely important for disease evolution.
General characteristics of the genome of Erwinia amylovora CFBP 1430 compared with E. amylovora Ea273 E. amylovora CFBP 1430 E. amylovora Ea273 
MAUVE alignment of the genome sequences of Erwinia amylovora CFBP 1430 and E. amylovora Ea273. Vertical lines indicate ribosomal (r)RNA operons. Numbers indicate the rRNA operon numerator, as used in the alignments. Arrows indicate the transcriptional direction of the rRNA operons. 
Fire blight, caused by the enterobacterium Erwinia amylovora, is a devastating disease of rosaceous plants that has global economic importance for apple and pear production and trade. The complete genome of E. amylovora CFBP 1430 was sequenced, annotated, and compared with the genomes of other Erwinia spp. Several singleton and shared features of the E. amylovora CFBP 1430 genome were identified that offer a first view into evolutionary aspects within the genus Erwinia. Comparative genomics identified or clarified virulence and fitness determinants and secretion systems. Novel insights revealed in the genome of E. amylovora CFBP 1430 hold potential for exploitation to improve the design of more effective fire blight control strategies.
Primers for SYBR Green real time polymerase chain reaction (PCR) experiments (sequences are 5′ to 3′) 
The ability of Pseudomonas syringae pv. phaseolicola to cause halo blight of bean is dependent on its ability to translocate effector proteins into host cells via the hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS). To identify genes encoding type III effectors and other potential virulence factors that are regulated by the HrpL alternative sigma factor, we used a hidden Markov model, weight matrix model, and type III targeting-associated patterns to search the genome of P. syringae pv. phaseolicola 1448A, which recently was sequenced to completion. We identified 44 high-probability putative Hrp promoters upstream of genes encoding the core T3SS machinery, 27 candidate effectors and related T3SS substrates, and 10 factors unrelated to the Hrp system. The expression of 13 of these candidate HrpL regulon genes was analyzed by real-time polymerase chain reaction, and all were found to be upregulated by HrpL. Six of the candidate type III effectors were assayed for T3SS-dependent translocation into plant cells using the Bordetella pertussis calmodulin-dependent adenylate cyclase (Cya) translocation reporter, and all were translocated. PSPPH1855 (ApbE-family protein) and PSPPH3759 (alcohol dehydrogenase) have no apparent T3SS-related function; however, they do have homologs in the model strain P. syringae pv. tomato DC3000 (PSPTO2105 and PSPTO0834, respectively) that are similarly upregulated by HrpL. Mutations were constructed in the DC3000 homologs and found to reduce bacterial growth in host Arabidopsis leaves. These results establish the utility of the bioinformatic or candidate gene approach to identifying effectors and other genes relevant to pathogenesis in P. syringae genomes.
Autoradiograph of p29, p38F2, p116, p149, and p174 DNA hybridizing to genomic DNA isolated from Pseudomonas syringae pv. syringae strains, digested with Dra I, and separated using clamped homogeneous electric field pulsed-field gel electrophoresis. Lane A, B301DR (parental); lane B, BR334 ( syrC mutant); lane C, BR334.5; lane D, BR334.1; lane E, BR334.3; lane F, BR334.2; lane G, BR334.5; and lane H, BR334.6. 
Diagrammatic representation of the approximately 145-kb Dra I fragment of Pseudomonas syringae pv. syringae strain B301D. The syringopeptin (red arrow) and syringomycin (blue arrow) gene clusters are presented with their relative location on the approximately 145-kb Dra I fragment. The position and orientation of the syrD , syrP , syrB1 , syrB2 , syrC , syrE , and sypA genes as organized on the P. syringae pv. syringae B301D chromosome are inset above the map of the approximately 145-kb Dra I fragment. The placement of the cosmids and subgenomic clones along the Dra I fragment are indicated below the map as solid black and diagonally hatched lines, respectively. The position of the Tn 3 HoHo1 insert 334 located within syrC is designated with a horizontal dotted line. Vertical arrows numbered 1 through 6 indicate the positions of the mini-Tn 5 insertions used to map the Dra I fragment. The phenotypic results of insertions along the 145-kb Dra I fragment are designated with a blue flag pointed right for loss of syringomycin production; a red flag pointed left for loss of syringopeptin production; and a double-sided black flag for loss of syringomycin and syringopeptin production. The restriction enzyme sites for Eco RI (E), Hin dIII (H), Kpn I (K), and Dra I are identified. 
Evaluation of the mini-Tn5 insertions in Pseudomonas syringae pv. syringae strain B310D, resultant phenotypes, and sequence analysis of the flanking DNA sequence
Genetic and phenotypic mapping of an approximately 145-kb DraI fragment of Pseudomonas syringae pv. syringae strain B301D determined that the syringomycin (syr) and syringopeptin (syp) gene clusters are localized to this fragment. The syr and syp gene clusters encompass approximately 55 kb and approximately 80 kb, respectively. Both phytotoxins are synthesized by a thiotemplate mechanism of biosynthesis, requiring large multienzymatic proteins called peptide synthetases. Genes encoding peptide synthetases were identified within the syr and syp gene clusters, accounting for 90% of the DraI fragment. In addition, genes encoding regulatory and secretion proteins were localized to the DraI fragment. In particular, the salA gene, encoding a regulatory element responsible for syringomycin production and lesion formation in P. syringae pv. syringae strain B728a, was localized to the syr gene cluster. A putative ATP-binding cassette (ABC) transporter homolog was determined to be physically located in the syp gene cluster, but phenotypically affects production of both phytotoxins. Preliminary size estimates of the syr and syp gene clusters indicate that they represent two of the largest nonribosomal peptide synthetase gene clusters. Together, the syr and syp gene clusters encompass approximately 135 kb of DNA and may represent a genomic island in P. syringae pv. syringae that contributes to virulence in plant hosts.
The biocontrol bacterium Paenibacillus alvei K165 has the ability to protect Arabidopsis thaliana against Verticillium dahliae. A direct antagonistic action of strain K165 against V. dahliae was ruled out, making it likely that K165-mediated protection results from induced systemic resistance (ISR) in the host. K165-mediated protection was tested in various Arabidopsis mutants and transgenic plants impaired in defense signaling pathways, including NahG (transgenic line degrading salicylic acid [SA]), etr1-1 (insensitive to ethylene), jar1-1 (insensitive to jasmonate), npr1-1 (nonexpressing NPR1 protein), pad3-1 (phytoalexin deficient), pad4-1 (phytoalexin deficient), eds5/sid1 (enhanced disease susceptibility), and sid2 (SA-induction deficient). ISR was blocked in Arabidopsis mutants npr1-1, eds5/sid1, and sid2, indicating that components of the pathway from isochorismate and a functional NPR1 play a crucial role in the K165-mediated ISR. Furthermore, the concomitant activation and increased transient accumulation of the PR-1, PR-2, and PR-5 genes were observed in the treatment in which both the inducing bacterial strain and the challenging pathogen were present in the rhizosphere of the A. thaliana plants.
Top-cited authors
Ben Lugtenberg
  • Leiden University
Corné Pieterse
  • Utrecht University
Guido V Bloemberg
  • Unilabs, Dübendorf, Switzerland
Sophien Kamoun
  • The Sainsbury Laboratory
Pugin Alain
  • French National Centre for Scientific Research