Initiation of plant disease resistance by physical interaction of AvrPto and Pto kinase

Science

Volumn 274, Issue 5295, 1996, Pages 2060-2063

  Tang, Xiaoyan ^a   Frederick, Reid D ^a   Zhou, Jianmin ^a   Halterman, Dennis A ^a   Jia, Yulin ^a   Martin, Gregory B ^a  

^a PURDUE UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

AGRICULTURE; ARTICLE; BACTERIAL VIRULENCE; DISEASE RESISTANCE; NONHUMAN; PRIORITY JOURNAL; PROTEIN PROTEIN INTERACTION; PSEUDOMONAS;

BACTERIA (MICROORGANISMS); LYCOPERSICON ESCULENTUM; PSEUDOMONAS;

EID: 0030448263     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5295.2060     Document Type: Article

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28. Oligonucleotide primers containing restriction enzyme sites were designed from conserved regions of the Pto and Fen genes that allowed the three carboxy-terminal regions to be amplified by PCR (22). PCR products were cleaved with appropriate restriction enzymes and ligated together to create chimeric Pto-Fen constructs (22). A conserved Bgl ll site in both Pto and Fen allowed for reciprocal amino terminal exchanges at amino acid 129. All constructs were verified by sequencing.
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30. Chimeric Pto-Fen gene constructs were cloned into pEG202 with the use of either Eco RI (Fen carboxyterminal region) or Eco RI-Bam HI (Pto carboxyl terminal region) sites and introduced into yeast EGY48 containing the avrPto gene in pJG4-5 (19).
31. Chimeric Pto-Fen gene constructs were cloned into pBl121 and the plasmids were introduced into A. tumefaciens EHA105. Chimeric constructs were transferred into tomato cultivar Moneymaker with the use of Agrobacterium-mediated transformation. Transgenic status of plants was verified by probing genomic DNA blots with a Pto gene probe and with the nptll gene from pBl121 (22).
32. Full-length avrPto and the avrPto deletions were ligated into the Eco RI and Xho I sites of pJG4-5 (19). Constructs were introduced into the yeast strain EGY48 containing the Pto gene in pEG202 (19). All constructs were verified by sequencing.
33. avrPto deletions were cloned into pPtE6 (11) and introduced into P. syringae tomato T1 or P. syringae tabaci 11528R by triparental mating. Transconjugants were verified by DNA blot analysis.
34. The reduced avirulence activity of the mutant AvrPto proteins might be due to less efficient secretion of AvrPto from the Pseudomonas cell. To examine this, avrPto deletions CA25, CΔ41, and CA74 were placed into pBl121 and tested with the Agrobacterium transient assay. Agrobacterium EHA105 containing avrPto induced an HR in 2 days, whereas EHA105 containing the avrPto deletion CΔ25 induced an HR after 4 days; the other deletions did not elicit an HR (X. Tang and G. Martin, unpublished results). This suggests that the carboxyl terminal 25 amino acids of AvrPto are not required for secretion from the bacterial cell; this portion of AvrPto may serve as an activation domain, interact with other components in the signaling pathway, or have a role in AvrPto stability.
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41. Levels of protein expression were determined with the use of antibody to LexA (a gift from E. Golemis) for LexA fusion proteins (in pEG202) and antibody to the hemagglutinin (HA) epitope tag (Boehringer-Manheim) for the AvrPto::HA fusion protein (in pJG4-5).
42. We thank L. Dunkle, A. Friedman, S. Gelvin, and K. Perry for helpful comments on the manuscript. Supported, in part, by a Purdue Research Foundation Doctoral Fellowship (D.H.), National Science Foundation grant MCB-93-03359 (G.B.M.) and a David and Lucile Packard Foundation Fellowship (G.B.M.).