Proc. Nati. Acad. Sci. USA Vol. 86, pp. 157-161, January 1989 Botany Cloned avirulence genes from the tomato pathogen Pseudomonas syringae pv. tomato confer cultivar specificity on soybean (host-parasite interactions/hypersensitive response/disease resistance/incompatibility) DONALD Y. KOBAYASHI, STANLEY J. TAMAKI, AND NOEL T. KEEN* Department of Plant Pathology, University of California, Riverside, CA 92521 Communicated by George A. Zentmyer, September 22, 1988 ABSTRACT Three different cosmid clones were isolated ever, avirulence genes have been cloned recently from from a genomic library of the tomato pathogen Pseudomonas several bacterial pathogens. syringae pv. tomato, which, when introduced into the soybean Staskawicz et al. (6) first cloned a gene for avirulence from pathogen P. syringae pv. glycinea, caused a defensive hyper- Pseudomonas syringae pv. glycinea race 6, the causal agent sensitive response (HR) in certain soybean cultivars. Each clone ofbacterial blight ofsoybean. This gene, designated avrA (7), was distinguished by the specific cultivars that reacted hyper- defines the P. syringae pv. glycinea race 6 phenotype and sensitively and by the intensity of the HR elicited. Unlike restricts the number of soybean cultivars that the pathogen wild-type P. syringae pv. tomato isolates, which elicit the HR on can attack. Additional avirulence genes were subsequently all soybean cultivars, all three clones exhibited cultivar spec- identified in other P. syringae pv. glycinea races (8) as well ificities analogous to avirulence genes previously cloned from P. as in pathovars ofXanthomonas campestris (9, 10). In all of these cases, avirulence genes have been shown to determine syringae pv. glycinea. However, the collective phenotypes ofthe the race phenotype of the pathogens, defined according to three clones accounted for HRs on all tested soybean cultivars. their range of virulence on a standard set of host cultivars, One of the three P. syringae pv. tomato clones contained an and therefore result in cultivar level resistance. avirulence gene homologous to avrA, which was previously In addition to race-cultivar specificity, higher orders of cloned from P. syringae pv. glycinea race 6. The other two P. plant-pathogen specificity occur. Pathogen subdivisions syringae pv. tomato clones expressed unique HR patterns on such as pathovars or formae speciales are defined largely various soybean cultivars, which were unlike those caused by according to their specific plant-species host range. In the any known P. syringae pv. glycinea race or previously cloned bacterial species P. syringae, there are >40 different patho- P. syringae pv. glycinea avr gene. Further characterization of vars specialized on a number ofdifferent hosts (11). Although the second P. syringae pv. tomato clone indicated that the the genetic factors conferring host-range determination avirulence phenotype resided on a 5.6-kilobase Hind]II frag- within this group are currently unknown, many P. syringae ment that, in Southern blot analyses, hybridized to an identical- pathovars as well as other plant pathogens frequently elicit size fragment in various P. syringae pathovars, including all the defensive HR on nonhost plant species (3). However, it tested glycinea races. These results demonstrate that avirulence has not been established whether these HRs causally limit the genes may be distributed among several P. syringae pathovars pathogen host range. In addition, the difficulty of intercross- but may be modified so that the HR is not elicited in a particular ing most plant species has hindered understanding the genetic host plant. Furthermore, the data raise the possibility that basis of host species resistance to pathogens. avirulence genes may function in host-range determination at The demonstration that avirulence genes may determine levels above race-cultivar specificity. race-cultivar specificity in several bacterial plant pathogens raised the possibility that higher levels of host-pathogen Disease resistance in plants frequently results from a basic specificity such as pathovar-plant species interactions may, incompatibility between the host and invading pathogen that in some cases, also be determined by pathogen avirulence is established by specific genetic factors in both organisms genes and corresponding plant disease-resistance genes. To (1). These factors appear to determine plant recognition of test this hypothesis, Pseudomonas syringae pv. tomato, the the pathogen during the early stages of infection, resulting in causal agent of bacterial speck of tomato, was chosen to a series of biochemical events in the plant that constitute a study the genetic factors involved in eliciting the HR on the localized defense mechanism called the hypersensitive re- nonhost plant, soybean. We report here, as in preliminary sponse (HR) (2). The HR is characterized by rapid necrosis reports (12, 13), the cloning and partial characterization of ofplant cells in proximity to the invading pathogen, followed three different avirulence genes from P. syringae pv. tomato by the accumulation of antimicrobial compounds termed that, when expressed in the soybean pathogen P. syringae phytoalexins as well as other factors (3). pv. glycinea, elicit the HR on soybean in a race-specific Genetic studies of both plants and pathogens have estab- manner. lished a gene-for-gene relationship in which elicitation of the HR requires a single dominant allele for resistance in the host cultivar and a complementary dominant gene for avirulence MATERIALS AND METHODS in the infecting pathogen race (1, 4). If either dominant allele Bacterial Strains, Vectors, Culture Media, and Antibiotics. is absent from the genotypes ofthe interacting organisms, the P. syringae strains, Escherichia coli strains, and plasmid defense response is not activated, and the plant is therefore vectors used in this study are listed in Table 1. E. coli cells susceptible to pathogen attack. Despite recent progress (for were routinely maintained on LB agar (14) and grown at 37°C. example, see ref. 5), disease-resistance genes have not yet P. syringae strains were maintained on KMB agar (19) at been cloned and characterized from any higher plant. How- 28°C. Antibiotics purchased from Sigma were used at the following levels unless otherwise indicated: tetracycline at The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: HR, hypersensitive response. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 157 Downloaded by guest on September 28, 2021 158 Botany: Kobayashi et al. Proc. Natl. Acad. Sci. USA 86 (1989) Table 1. Bacterial strains and plasmids Relevant characteristics or geographic origin Source (ref.) Strain E. coli HB101 F-hsdS20 [hsdR hsdM recA13 ara-14 proA2 lacYI galK2 rpsL20 (Strr) (14) xyl-S mtl-I supE44 A-] DH5a F-lacZ AM15 endAl recAl hsdR17 supE44 thi-1 gyrA relAl A- Bethesda Research Laboratories P. syringae pv. glycinea race 0 B. Staskawicz (8) race 4 Rifr This lab race 5 Rifr This lab race 6 Rifr This lab pv. lachrymans This lab pv. mori UCPPB 566 D. A. Cooksey pv. phaseolicola 0285-1 D. A. Cooksey pv. maculicola 1083-2 D. A. Cooksey pv. tomato PT20 Imperial Valley, CA D. A. Cooksey (15) PT23 San Diego, CA D. A. Cooksey (15) P.T.F. Michigan D. A. Cooksey (15) P144 New Jersey D. A. Cooksey PT178 North Carolina D. A. Cooksey B120 Delaware T. Denny Plasmid pLAFR3 Tcr, cosmid derivative of RK2 (8) pRK2013 Kmr, Tra, helper plasmid derivative from RK2 (16) pRK415 Tcr, RK2 plasmid derivative (17) pUC118 Apr, pUC18 derivative (18) pPSG6001 3.2-kb Acc I fragment containing avrA from P. syringae pv. glycinea (7) race 6 in pUC8 pPrT4D2 Class I cosmid clone This work pPT7H6 Class I cosmid clone This work pP1 9A11 Class I cosmid clone This work pPT201 3.2-kb Sal I fragment subclone from pPT9A11 in pRK415 This work pPT211 3.2-kb Sal I fragment subclone from pPT9A11 in pUC118 This work pPI4E1O Class II cosmid clone This work pPR101 5.6-kb HindIl fragment from pPT4E10 in pRK415 This work pPT112 pPT101 with TnS insert mutating avirulence gene activity This work pPTlOE9 Class III cosmid clone This work Ap, Ampicillin; Km, kanamycin; Rif, rifampicin; Tc, tetracycline; r, resistant. 12.5 ug/ml, kanamycin at 50 ,ug/ml, and rifampicin at 100 25 ug/ml. Single colonies were selected and successively ,ug/ml. transferred on selective medium to make up the inoculum, Recombinant DNA Techniques and Cosmid Library Con- which was prepared to a final concentration of 107 cells per struction. Methods for plasmid DNA isolations, restriction ml by suspending cells in sterile water. digests, ligations, Southern DNA transfers, and nick-trans- Growth conditions for plants are described elsewhere (20). lations were performed as described by Maniatis et al. (14). Cell suspensions were infiltrated into the primary leaves of Isolation of total DNA from P. syringae pv. tomato PT23 10-day-old soybeans by using a Hagborg device (21). was performed as described by Staskawicz et al. (6), and TnS Mutagenesis of Cosmid Clones. TnS mutagenesis ofthe construction of a genomic library in the cosmid vector cosmid clone pPT4E10 was conducted in E. coli DH5a by pLAFR3 was carried out as described (8). Cosmids were using phage A::TnS as described (22). packaged in phage extracts purchased from Boehringer Mannheim. RESULTS For Southern blot analyses, 1 ug of plasmid DNA or 4 ,ug P. syringae pv. tomato Library Construction and Identifica- of total genomic DNA was digested with the appropriate tion of Cosmid Clones. Transduction of E. coli HB101 with restriction enzymes and electrophoresed in 0.7% agarose packaged cosmid DNA yielded >104 tetracycline-resistant before transfer onto Zetabind nylon membranes (AMF). colonies per ,ug of DNA. One thousand colonies were Hybridizations with 32P-labeled probes were carried out at selected to comprise the library, of which 25 clones were 42°C in 50% (wt/vol) formamide/0.75 M NaCl/0.075 M randomly selected for plasmid DNA analysis.