Functional analysis of the early chlorosis factor gene.

Chlorosis is one of the symptoms of bacterial spot disease caused by Xanthomonas campestris pv. vesicatoria, which induces chlorosis before any other symptoms appear on tomato. We report characterization of a 2.1-kb gene called early chlorosis factor (ecf). The gene ecf encodes a hydrophobic protein with similarity to four other proteins in plant pathogens, including HolPsyAE, and uncharacterized gene products from X. campestris pv. campestris and X. axonopodis pv. citri, and, at the tertiary structure level, to colicin Ia from Escherichia coli. We demonstrate that the associated phenotype is hrp dependent, and that the ecf gene product appears to be translocated to host cells. The gene ecf has no impact on electrolyte leakage or on bacterial growth in planta in response to infection. Concentrated culture filtrates do not produce chlorosis. Study of its role in Xanthomonas spp.-tomato interactions will forward our understanding of symptom production by plant pathogens and allows further investigation into the mechanisms of bacterial virulence and production of symptoms.

Avirulence gene avrRxv from Xanthomonas campestris pv. vesicatoria specifies resistance on tomato line Hawaii 7998.

The molecular and genetic control of the interaction between tomato races of Xanthomonas campestris pv. vesicatoria (XcvT) and tomato was studied. Based on inoculation phenotype and analysis of in planta bacterial growth, tomato line Hawaii 7998 is resistant to XcvT race 1 75-3 but not to XcvT race 2 89-1. Two cosmid clones from a genomic library of XcvT race 1 75-3 converted the normally virulent race 2 89-1 to avirulence on Hawaii 7998. The two clones contained the previously isolated, nonhost avirulence gene avrRxv, and their activity was localized to a 2.1-kbp subclone of avrRxv. avrRxv inhibits growth of race 2 89-1 in the resistant line Hawaii 7998 and an insertional mutation in avrRxv prevents this inhibition. In addition, a dramatic increase in electrolyte leakage of leaves of Hawaii 7998 occurred after 12-hr postinfiltration with race 2 89-1 carrying avrRxv. The nucleotide sequence of avrRxv revealed one major open reading frame (ORF) that accords well with activity analysis of nested deletions. ORF 2-2 encodes a putative protein of 374 amino acids with a molecular weight of 42.1 kDa and a pI of 10.7. Inheritance of the avrRxv-specific resistance in Hawaii 7998 was studied in a total of 587 F2 individuals from crosses between Hawaii 7998 and susceptible lines. The inheritance of avrRxv-specific resistance in Hawaii 7998 appears to be governed by more than one locus.

Regulation of expression of avirulence gene avrRxv and identification of a family of host interaction factors by sequence analysis of avrBsT.

Resistance in tomato line Hawaii 7998 as well as in several nonhost plants to Xanthomonas campestris pv. vesicatoria tomato strain (XcvT) is mediated in part by the avirulence gene avrRxv. Analysis of growth of wild-type and avrRxv deletion strains indicates that avrRxv plays a crucial role in the ability of XcvT 92-14 to induce resistance on Hawaii 7998. We used avrRxv reporter gene fusions and Northern (RNA) blot analysis to test several growth environments for inductive potential. We found that avrRxv is constitutively expressed at high levels and that growth in planta, in tobacco conditioned medium, and in hrp-inductive medium XVM2 did not affect the high levels of expression. In addition, hrp structural and regulatory mutant backgrounds had no effect. We mutated the bipartite plant inducible promoter (PIP)-box sequence and found that avrRxv activity appears to be independent of an intact PIP-box element. We present the sequence of the avrRxv homologue called avrBsT and align the six AvrRxv host interaction factor family members including mammalian pathogen virulence factors YopJ and YopP from Yersinia spp. and AvrA from Salmonella typhimurium, and open reading frame Y4LO with unknown function from the symbiont Rhizobium sp.

The effect of ethylene on root growth of Zea mays seedlings.

The control of primary root growth in Zea mays cv. Merit by ethylene was examined. At applied concentrations of ethylene equal to or greater than 0.1 microliter L-1, root elongation during 24 h was inhibited. The half-maximal response occurred at 0.6 microliter L-1 and the response saturated at 6 microliters L-1. Inhibition of elongation took place within 20 min. However, after ethylene was removed, elongation recovered to control values within 15 min. Root elongation was also inhibited by green light. The inhibition caused by a 24-h exposure to ethylene was restricted to the elongating region just behind the apex, with inhibition of cortical cell elongation being the primary contributor to the effect. Based on use of 2,5-norbornadiene, a gaseous competitive inhibitor of ethylene, it was concluded that endogenous ethylene normally inhibits root elongation.

Characterization of a gene from a tomato pathogen determining hypersensitive resistance in non-host species and genetic analysis of this resistance in bean.

Xanthomonas campestris pv. vesicatoria is the causal agent of leaf spot disease on pepper and tomato. On non-host plants, such as bean, soybean, cowpea, alfalfa, and cotton, X. campestris pv. vesicatoria is unable to cause disease, inducing instead a hypersensitive resistance response (HR). Since avirulence genes from X. campestris pv. vesicatoria specifically induce HR in several pepper cultivars, we investigated whether there were avirulence genes governing induction of resistance in non-host species. We report on the molecular cloning and characterization of a non-host avirulence gene from X. campestris pv. vesicatoria. A cosmid clone isolated from a library of DNA from X. campestris pv. vesicatoria tomato race 1 converted X. campestris pv. phaseoli to avirulence by inducing HR on the bean cultivar Sprite, but not on Bush Blue Lake. The HR-inducing activity was localized to a 2.1-kilobase Pst I fragment of DNA, designated avrRxv. In addition, we demonstrate that avrRxv inhibited disease production by several X. campestris pathovars on their normally susceptible hosts: glycines on soybean, vignicola on cowpea, alfalfae on alfalfa, holcicola on corn, and malvacearum on cotton. The HR resistance in bean induced by avrRxv segregated as a single incompletely dominant gene, designated Rxv. These results indicate that the avirulence gene avrRxv and the resistance gene Rxv partially control the outcome of the interaction between X. campestris pv. vesicatoria and the non-host bean.

Identification of Pseudomonas syringae pathogens of Arabidopsis and a bacterial locus determining avirulence on both Arabidopsis and soybean.

To develop a model system for molecular genetic analysis of plant-pathogen interactions, we studied the interaction between Arabidopsis thaliana and the bacterial pathogen Pseudomonas syringae pv tomato (Pst). Pst strains were found to be virulent or avirulent on specific Arabidopsis ecotypes, and single ecotypes were resistant to some Pst strains and susceptible to others. In many plant-pathogen interactions, disease resistance is controlled by the simultaneous presence of single plant resistance genes and single pathogen avirulence genes. Therefore, we tested whether avirulence genes in Pst controlled induction of resistance in Arabidopsis. Cosmids that determine avirulence were isolated from Pst genomic libraries, and the Pst avirulence locus avrRpt2 was defined. This allowed us to construct pathogens that differed only by the presence or absence of a single putative avirulence gene. We found that Arabidopsis ecotype Col-0 was susceptible to Pst strain DC3000 but resistant to the same strain carrying avrRpt2, suggesting that a single locus in Col-0 determines resistance. As a first step toward genetically mapping the postulated resistance locus, an ecotype susceptible to infection by DC3000 carrying avrRpt2 was identified. The avrRpt2 locus from Pst was also moved into virulent strains of the soybean pathogen P. syringae pv glycinea to test whether this locus could determine avirulence on soybean. The resulting strains induced a resistant response in a cultivar-specific manner, suggesting that similar resistance mechanisms may function in Arabidopsis and soybean.

Expression of the Bs2 pepper gene confers resistance to bacterial spot disease in tomato.

The Bs2 resistance gene of pepper specifically recognizes and confers resistance to strains of Xanthomonas campestris pv. vesicatoria that contain the corresponding bacterial avirulence gene, avrBs2. The involvement of avrBs2 in pathogen fitness and its prevalence in many X. campestris pathovars suggests that the Bs2 gene may be durable in the field and provide resistance when introduced into other plant species. Employing a positional cloning strategy, the Bs2 locus was isolated and the gene was identified by coexpression with avrBs2 in an Agrobacterium-mediated transient assay. A single candidate gene, predicted to encode motifs characteristic of the nucleotide binding site-leucine-rich repeat class of resistance genes, was identified. This gene specifically controlled the hypersensitive response when transiently expressed in susceptible pepper and tomato lines and in a nonhost species, Nicotiana benthamiana, and was designated as Bs2. Functional expression of Bs2 in stable transgenic tomatoes supports its use as a source of resistance in other Solanaceous plant species.

Short communication: developmental control of Xa21-mediated disease resistance in rice.

The rice resistance gene Xa21 confers resistance against the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). The molecular genetic mechanism controlling the integration of the Xa21-mediated disease resistance response with the developmental program in rice is under study in this model system. Reproducible means of infecting plants at certain developmental stages were designed based on the timing of full expansion of the leaf. Xa21-resistance progressively increases from the susceptible juvenile leaf 2 stage through later stages, with 100% resistance at the adult leaf 9/10 stage. We found that Xa21 expression is independent of plant developmental stage, infection with Xoo, or wounding. Expression of the Xa21 gene transcript is not correlated with expression of Xa21 disease resistance indicating that the developmental regulation of Xa21-resistance is either controlled post-transcriptionally or by other factors.

Xa21D encodes a receptor-like molecule with a leucine-rich repeat domain that determines race-specific recognition and is subject to adaptive evolution.

The rice Xa21 gene confers resistance to Xanthomonas oryzae pv oryzae in a race-specific manner. Analysis of the inheritance patterns and resistance spectra of transgenic plants carrying six Xa21 gene family members indicated that one member, designated Xa21D, displayed a resistance spectrum identical to that observed for Xa21 but conferred only partial resistance. Xa21D encodes a receptor-like protein carrying leucine-rich repeat (LRR) motifs in the presumed extracellular domain. The Xa21D transcript terminates shortly after the stop codon introduced by the retrotransposon Retrofit. Comparison of nucleotide substitutions in the LRR coding regions of Xa21 and Xa21D provided evidence of adaptive selection. Both functional and evolutionary evidence indicates that the Xa21D LRR domain controls race-specific pathogen recognition.