hxc2, an Arabidopsis mutant with an altered hypersensitive response to Xanthomonas campestris pv. campestris

A chemical mutagenized population of Arabidopsis Col-0-gl plants was screened for an altered hypersensitive response (HR) after spray inoculation with an HR-inducing isolate of Xanthomonas campestris pv. campestris (strain 147). Three classes of mutant were identified: those exhibiting an HR- phenotype or partial loss of HR; hyper-responsive mutants showing necrotic lesions rapidly leading to the collapse of leaves; and susceptible mutants. One mutant belonging to the susceptible class, hxc-2, was extensively characterized. The compatible phenotype observed several days after initiation of the interaction was confirmed by measurement of in planta bacterial growth and use of bacterial strains constitutively expressing the GUS reporter gene. In the same way, accumulation of autofluorescent compounds, salicylic acid production and defence gene expression in the mutant were found to be similar to that displayed by the susceptible ecotype. Inoculation of hxc-2 with different avirulent bacteria suggests that the mutation is specific for the interaction with the Xcc 147 strain, although the mutation has been shown to affect a single dominant locus, different from the resistance locus defined by genetic analysis of resistance to Xcc 147. Genetic mapping of the mutation indicated that it is located on chromosome III, defining a previously unknown resistance function in response to X. c. campestris.     

The type III effector AvrXccB in Xanthomonas campestris pv. campestris targets putative methyltransferases and suppresses innate immunity in Arabidopsis

Xanthomonas campestris pv. campestris (Xcc) causes black rot, one of the most important diseases of brassica crops worldwide. The type III effector inventory plays important roles in the virulence and pathogenicity of the pathogen. However, little is known about the virulence function(s) of the putative type III effector AvrXccB in Xcc. Here, we investigated the immune suppression ability of AvrXccB and the possible underlying mechanisms. AvrXccB was demonstrated to be secreted in a type III secretion system‐dependent manner. AvrXccB tagged with green fluorescent protein is localized to the plasma membrane in Arabidopsis, and the putative N‐myristoylation motif is essential for its localization. Chemical‐induced expression of AvrXccB suppresses flg22‐triggered callose deposition and the oxidative burst, and promotes the in planta growth of Xcc and Pseudomonas syringae pv. tomato in transgenic Arabidopsis plants. The putative catalytic triad and plasma membrane localization of AvrXccB are required for its immunosuppressive activity. Furthermore, it was demonstrated that AvrXccB interacts with the Arabidopsis S‐adenosyl‐l ‐methionine‐dependent methyltransferases SAM‐MT1 and SAM‐MT2. Interestingly, SAM‐MT1 is not only self‐associated, but also associated with SAM‐MT2 in vivo. SAM‐MT1 and SAM‐MT2 expression is significantly induced upon stimulation of microbe‐associated molecular patterns and bacterial infection. Collectively, these findings indicate that AvrXccB targets a putative methyltransferase complex and suppresses plant immunity.     

Comparative microscopic and enzymatic characterization of the leaf necrosis induced in Arabidopsis thaliana by lead nitrate and by Xanthomonas campestris pv. campestris after foliar spray

The heavy metal lead was administered to Arabidopsis thaliana plants by foliar spray. At a concentration of l4mol m^?3, the lead nitrate suspension induced densely distributed necrotic lesions on A. thaliana leaves. A number of Arabidopsis ecotypes were tested and a differential response to heavy-metal toxicity was noted. The necrosis provoked as a result of the phytotoxic effect of lead had a similar appearance to the necrotic lesions observed in a hypersensitive response of A. thaliana to inoculation with Xanthomonas campestris pv. campestris (Lummerzheim et al. 1993, Molecular Plant-Microbe Interactions 6, 532–544). In addition to this phenotypic resemblance, accumulation of polyphenols and callose depositions observed by microscopic analysis, as well as increases in the activities of the stress-related proteins β1,3-glucanases, chitinases and peroxidases, revealed significant similarities in the plant response to the two treatments examined, lead toxicity and bacterial infection. The results allow the establishment of markers for both types of stress.     

Antisense inhibition of protein phosphatase 2C accelerates cold acclimation in Arabidopsis thaliana

Two related protein phosphatases 2C, ABI1 and AtPP2CA have been implicated as negative regulators of ABA signalling. In this study we characterized the role of AtPP2CA in cold acclimation. The pattern of expression of AtPP2CA and ABI1 was studied in different tissues and in response to abiotic stresses. The expression of both AtPP2CA and ABI1 was induced by low temperature, drought, high salt and ABA. The cold and drought-induced expression of these genes was ABA-dependent, but divergent in various ABA signalling mutants. In addition, the two PP2C genes exhibited differences in their tissue-specific expression as well as in temporal induction in response to low temperature. To elucidate the function of AtPP2CA in cold acclimation further, the corresponding gene was silenced by antisense inhibition. Transgenic antisense plants exhibited clearly accelerated development of freezing tolerance. Both exposure to low temperature and application of ABA resulted in enhanced freezing tolerance in antisense plants. These plants displayed increased sensitivity to ABA both during development of frost tolerance and during seed germination, but not in their drought responses. Furthermore, the expression of cold-and ABA-induced genes was enhanced in transgenic antisense plants. Our results suggest that AtPP2CA is a negative regulator of ABA responses during cold acclimation.     

The role of type III effectors from Xanthomonas axonopodis pv. manihotis in virulence and suppression of plant immunity

Xanthomonas axonopodis pv. manihotis (Xam) causes cassava bacterial blight, the most important bacterial disease of cassava. Xam, like other Xanthomonas species, requires type III effectors (T3Es) for maximal virulence. Xam strain CIO151 possesses 17 predicted T3Es belonging to the Xanthomonas outer protein (Xop) class. This work aimed to characterize nine Xop effectors present in Xam CIO151 for their role in virulence and modulation of plant immunity. Our findings demonstrate the importance of XopZ, XopX, XopAO1 and AvrBs2 for full virulence, as well as a redundant function in virulence between XopN and XopQ in susceptible cassava plants. We tested their role in pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI) and effector‐triggered immunity (ETI) using heterologous systems. AvrBs2, XopR and XopAO1 are capable of suppressing PTI. ETI suppression activity was only detected for XopE4 and XopAO1. These results demonstrate the overall importance and diversity in functions of major virulence effectors AvrBs2 and XopAO1 in Xam during cassava infection.     

Transgenic expression of the rice Xa21 pattern‐recognition receptor in banana (Musa sp.) confers resistance to Xanthomonas campestris pv. musacearum

Banana Xanthomonas wilt (BXW), caused by the bacterium Xanthomonas campestris pv. musacearum (Xcm), is the most devastating disease of banana in east and central Africa. The spread of BXW threatens the livelihood of millions of African farmers who depend on banana for food security and income. There are no commercial chemicals, biocontrol agents or resistant cultivars available to control BXW. Here, we take advantage of the robust resistance conferred by the rice pattern‐recognition receptor (PRR), XA21, to the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). We identified a set of genes required for activation of Xa21‐mediated immunity (rax) that were conserved in both Xoo and Xcm. Based on the conservation, we hypothesized that intergeneric transfer of Xa21 would confer resistance to Xcm. We evaluated 25 transgenic lines of the banana cultivar ‘Gonja manjaya’ (AAB) using a rapid bioassay and 12 transgenic lines in the glasshouse for resistance against Xcm. About 50% of the transgenic lines showed complete resistance to Xcm in both assays. In contrast, all of the nontransgenic control plants showed severe symptoms that progressed to complete wilting. These results indicate that the constitutive expression of the rice Xa21 gene in banana results in enhanced resistance against Xcm. Furthermore, this work demonstrates the feasibility of PRR gene transfer between monocotyledonous species and provides a valuable new tool for controlling the BXW pandemic of banana, a staple food for 100 million people in east Africa.     

Xanthomonas campestris pv. campestris lpsI and lpsJ genes encoding putative proteins with sequence similarity to the α- and β-subunits of 3-oxoacid CoA-transferases are involved in LPS biosynthesis

A 2.1-kb SmaI-EcoRI DNA fragment upstream of the xanA and xanB genes of Xanthomonas campestris pv. campestris carries two ORFs encoding putative proteins with sequence similarities to the α- and β-subunits of 3-oxoacid-CoA transferases. The two ORFs were termed lpsI and lpsJ because strains carrying appropriate mutations showed an autoagglutination phenotype and because lipopolysaccharides of these mutant strains were altered according to silver-stained polyacrylamide gels. The monosaccharide composition of the exopolysaccharide xanthan produced by lpsI and lpsJ mutants remained unchanged. Received: 29 March 1997 / Accepted: 21 July 1997     

Identification of novel HrpXo regulons preceded by two cis-acting elements, a plant-inducible promoter box and a -10 box-like sequence, from the genome database of Xanthomonas oryzae pv. oryzae

A regulatory protein HrpXo of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, is known to control the expression of hrp genes that encode components of a type III secretion system and of some effector protein genes. In this study, we screened novel HrpXo regulons from the genome database of X. oryzae pv. oryzae, searching for ORFs preceded by two predicted sequence motifs, a plant-inducible promoter box-like sequence and a -10 box-like sequence. Using a gus reporter system, nine of 15 ORF candidates were expressed HrpXo dependently. We also showed by base-substituted mutagenesis that both motifs are essential for the expression of the genes.     

The TIR-NBS protein TN13 associates with the CC-NBS-LRR resistance protein RPS5 and contributes to RPS5-triggered immunity in Arabidopsis

Nucleotide-binding site (NBS)–leucine-rich repeat (LRR) domain receptor (NLR) proteins play important roles in plant innate immunity by recognizing pathogen effectors. The Toll/interleukin-1 receptor (TIR)-NBS (TN) proteins belong to a subtype of the atypical NLRs, but their function in plant immunity is poorly understood. The well-characterized Arabidopsis thaliana typical coiled-coil (CC)-NBS-LRR (CNL) protein Resistance to Pseudomonas syringae 5 (RPS5) is activated after recognizing the Pseudomonas syringae type III effector AvrPphB. To explore whether the truncated TN proteins function in CNL-mediated immune signaling, we examined the interactions between the Arabidopsis TN proteins and RPS5, and found that TN13 and TN21 interacted with RPS5. However, only TN13, but not TN21, was involved in the resistance to P. syringae pv. tomato (Pto) strain DC3000 carrying avrPphB, encoding the cognate effector recognized by RPS5. Moreover, the regulation of Pto DC3000 avrPphB resistance by TN13 appeared to be specific, as loss of function of TN13 did not compromise resistance to Pto DC3000 hrcC⁻ or Pto DC3000 avrRpt2. In addition, we demonstrated that the CC and NBS domains of RPS5 play essential roles in the interaction between TN13 and RPS5. Taken together, our results uncover a direct functional link between TN13 and RPS5, suggesting that TN13 acts as a partner in modulating RPS5-activated immune signaling, which constitutes a previously unknown mechanism for TN-mediated regulation of plant immunity.     

Potential of doubled-haploid lines and localization of quantitative trait loci (QTL) for partial resistance to bacterial leaf streak (Xanthomonas campestris pv. hordei) in barley

 Genetic variability for partial resistance to bacterial leaf streak in barley, caused by Xanthomonas campestris pv. hordei, was investigated in 119 doubled-haploid lines (DH) developed by the Hordeum bulbosum method from the F₁ progeny of the cross between two cultivars, ‘Morex’ (resistant) and ‘Steptoe’ (susceptible). Two experiments were undertaken in a randomized complete block design with three replicates, in a controlled growth chamber. Twenty seeds per replicate were planted in plastic containers (60×40×8 cm) containing moistened vermiculite. At the two-leaf stage seedlings were inoculated with an Iranian strain of the pathogen. Genetic variability was observed among the 119 DH lines for partial resistance to the disease. Some DH lines were significantly more resistant than ‘Morex’ (resistant parent) to bacterial leaf streak. Genetic gain in percentage of resistant parent for 5% of the selected DH lines was significant (47.70% and 33.72% in the first and the second experiment, respectively). A QTL analysis of bacterial leaf streak resistance showed that three QTLs were detected on chromosomes 3 and 7. Multilocus allelic effects of the three QTLs account for almost 54% of the mean difference between the parents and nearly 30% of the phenotypic variation of the trait in the mean experiment. The resistance locus on chromosome 3, near ABG377, apprears to be a major gene. Received: 15 July 1997 / Accepted: 4 August 1997     

Arabidopsis Qa-SNARE SYP2 proteins localized to different subcellular regions function redundantly in vacuolar protein sorting and plant development

SYP2 proteins are a sub-family of Qa-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) that may be responsible for protein trafficking between pre-vacuolar compartments (PVC) and vacuoles. Arabidopsis thaliana SYP22/VAM3/SGR3 and SYP21/PEP12 proteins function independently, but are both reported to be essential for male gametophytic viability. Here, we systematically examined the redundancy of three SYP2 paralogs (i.e. SYP21, 22 and 23) using a Col-0 ecotype harboring a SYP2 paralog (SYP23/PLP) that lacked a transmembrane domain. Surprisingly, no visible phenotypes were observed, even in the double knockout syp21/pep12 syp23/plp. Deficiency of either SYP21/PEP12 or SYP23/PLP in the syp22 background resulted in a defect in vacuolar protein sorting, characterized by abnormal accumulation of protein precursors in seeds. SYP21/PEP12 knockdown enhanced the syp22 phenotype (i.e. semi-dwarfism, poor leaf vein development and abnormal development of myrosin cells), and additional knockout of SYP23/PLP further aggravated the phenotype. A GFP-SYP23/PLP fusion localized to the cytosol, but not to the PVC or vacuolar membrane, where SYP21/PEP12 or SYP22/VAM3, respectively, were localized. Immunoprecipitation analysis showed that SYP23/PLP interacted with the vacuolar Qb- and Qc-SNAREs, VTI11 and SYP5, respectively, suggesting that SYP23/PLP is able to form a SNARE complex anchoring the membrane. Unexpectedly, we found that expression of multiple copies of a genomic fragment of SYP23/PLP suppressed the abnormal syp22-3 phenotype. Thus, SYP2 proteins, including cytosolic SYP23/PLP, appear to function redundantly in vacuolar trafficking and plant development.     

IDL6‐HAE/HSL2 impacts pectin degradation and resistance to Pseudomonas syringae pv tomato DC3000 in Arabidopsis leaves

Plant cell walls undergo dynamic structural and chemical changes during plant development and growth. Floral organ abscission and lateral root emergence are both accompanied by cell‐wall remodeling, which involves the INFLORESCENCE DEFICIENT IN ABSCISSION (IDA)‐derived peptide and its receptors, HAESA (HAE) and HAESA‐LIKE2 (HSL2). Plant cell walls also act as barriers against pathogenic invaders. Thus, the cell‐wall remodeling during plant development could have an influence on plant resistance to phytopathogens. Here, we identified IDA‐like 6 (IDL6), a gene that is prominently expressed in Arabidopsis leaves. IDL6 expression in Arabidopsis leaves is significantly upregulated when the plant is suffering from attacks of the bacterial Pseudomonas syringae pv. tomato (Pst) DC3000. IDL6 overexpression and knockdown lines respectively decrease and increase the Arabidopsis resistance to Pst DC3000, indicating that the gene promotes the Arabidopsis susceptibility to Pst DC3000. Moreover, IDL6 promotes the expression of a polygalacturonase (PG) gene, ADPG2, and increases PG activity in Arabidopsis leaves, which in turn reduces leaf pectin content and leaf robustness. ADPG2 overexpression restrains Arabidopsis resistance to Pst DC3000, whereas ADPG2 loss‐of‐function mutants increase the resistance to the bacterium. Pst DC3000 infection elevates the ADPG2 expression partially through HAE and HSL2. Taken together, our results suggest that IDL6‐HAE/HSL2 facilitates the ingress of Pst DC3000 by promoting pectin degradation in Arabidopsis leaves, and Pst DC3000 might enhance its infection by manipulating the IDL6‐HAE/HSL2‐ADPG2 signaling pathway.     

HrpB7 from Xanthomonas campestris pv. vesicatoria is an essential component of the type III secretion system and shares features of HrpO/FliJ/YscO family members

The Gram‐negative bacterium Xanthomonas campestris pv. vesicatoria translocates effector proteins via a type III secretion system (T3SS) into eukaryotic cells. The T3SS spans both bacterial membranes and consists of more than 20 proteins, 9 of which are conserved in plant and animal pathogens and constitute the core subunits of the secretion apparatus. T3S in X. campestris pv. vesicatoria also depends on nonconserved proteins with yet unknown function including HrpB7, which contains predicted N‐ and C‐terminal coiled‐coil regions. In the present study, we provide experimental evidence that HrpB7 forms stable oligomeric complexes. Interaction and localisation studies suggest that HrpB7 interacts with inner membrane and predicted cytoplasmic (C) ring components of the T3SS but is dispensable for the assembly of the C ring. Additional interaction partners of HrpB7 include the cytoplasmic adenosinetriphosphatase HrcN and the T3S chaperone HpaB. The interaction of HrpB7 with T3SS components as well as complex formation by HrpB7 depends on the presence of leucine heptad motifs, which are part of the predicted N‐ and C‐terminal coiled‐coil structures. Our data suggest that HrpB7 forms multimeric complexes that associate with the T3SS and might serve as a docking site for the general T3S chaperone HpaB.