Molecular characterization of the vir regulon of Agrobacterium tumefaciens: Complete nucleotide sequence and gene organization of the 28.63-kbp regulon cloned as a single unit

Rogowsky, Peter; Powell, Bradford S.; Shirasu, Ken; Lin, Tzu‐Se; Morel, Patrice; Zyprian, Eva; Steck, Todd R.; Kado, Clarence I.

doi:10.1016/0147-619x(90)90028-b

Cited by 105 publications

(64 citation statements)

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“…(C) Nucleotide sequence of virG promoter region from two wild-type strains, C58 and D10B/87 (D10). The complete sequence is presented for strain C58 (18). For the wild-type strain D10B/87, only the nucleotide changes with respect to the C58 sequence are presented.…”

Section: Resultsmentioning

confidence: 99%

“…The complete virG sequence of strain D10B/87 has been submitted to GenBank under accession number U16142. The virG sequence of strain C58 has been deposited by Rogowsky et al in EMBL and GenBank data libraries under accession number J03320 (18).…”

Section: Methodsmentioning

confidence: 99%

“…The guanidium thiocyanate method was used for total RNA extraction and Northern dot blot analysis was performed under high stringency conditions (2). The 555-bp virG DNA fragment used as probe for Northern dot blot hybridizations covered nucleotides 14,678 to 15,232 of the C58 vir sequence (18). A 312-bp PCR fragment, generated from a conserved portion of 16S rRNA genes by using primers Y1 and Y2 (22) and Rhizobium meliloti A3 DNA, was used as a control probe to verify that equal amounts of RNA had been blotted.…”

Section: Methodsmentioning

confidence: 99%

“…pTiD10B/87 virG DNA was amplified by using PCR and oligonucleotide primers complementary to the pTiC58 sequence corresponding to positions 14,387 to 14,407 and 15,520 to 15,540 of the vir region (18). The virG PCR product was purified with Magic PCR Prep (Promega) before sequencing with the AmpliTaq cycle sequencing kit (Perkin-Elmer Cetus).…”

Section: Methodsmentioning

confidence: 99%

“…A 312-bp PCR fragment, generated from a conserved portion of 16S rRNA genes by using primers Y1 and Y2 (22) and Rhizobium meliloti A3 DNA, was used as a control probe to verify that equal amounts of RNA had been blotted. Primer extension (2) was carried out by using total RNA from acetosyringone-treated bacteria and primer ϩ40Ϫ, corresponding to positions 14,747 to 14,767 of the vir region (18).…”

Section: Methodsmentioning

confidence: 99%

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Genetic analysis of nonpathogenic Agrobacterium tumefaciens mutants arising in crown gall tumors

et al. 1995

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Little is known about the effect of the host on the genetic stability of bacterial plant pathogens. Crown gall, a plant disease caused by Agrobacterium tumefaciens, may represent a useful model to study this effect. Indeed, our previous observations on the natural occurrence and origin of nonpathogenic agrobacteria suggest that the host plant might induce loss of pathogenicity in populations of A. tumefaciens. Here we report that five different A. tumefaciens strains initially isolated from apple tumors produced up to 99% nonpathogenic mutants following their reintroduction into axenic apple plants. Two of these five strains were also found to produce mutants on pear and/or blackberry plants. Generally, the mutants of the apple isolate D10B/87 were altered in the tumor-inducing plasmid, harboring either deletions in this plasmid or point mutations in the regulatory virulence gene virG. Most of the mutants originating from the same tumor appeared to be of clonal origin, implying that the host plants influenced agrobacterial populations by favoring growth of nonpathogenic mutants over that of wild-type cells. This hypothesis was confirmed by coinoculation of apple rootstocks with strain D10B/87 and a nonpathogenic mutant.The virulence (vir) and transferred (T) regions of Agrobacterium tumefaciens Ti plasmids contain genes directly involved in plant transformation. Expression of the vir genes by the bacterium is induced through the action of the virA-virG twocomponent regulatory system in response to plant phenolic compounds such as acetosyringone. The T-DNA, produced from the T region at the direction of the vir genes, becomes integrated into plant chromosomal DNA, where it determines the synthesis of plant growth hormones responsible for crown gall tumor development. The T-DNA also confers on tumor cells the capacity to produce unusual metabolites called opines. One of these opines, nopaline, is synthesized through the reductive condensation of arginine and ␣-ketoglutaric acid. Other Ti plasmid-encoded genes, which are not transferred to plant cells, enable the bacterial pathogen to utilize opines for growth and tumor colonization (for reviews, see references 5, 6, and 20). In spite of the enhanced colonization potential associated with expression of the opine catabolic genes, pathogenic Agrobacterium strains are difficult to recover from certain types of tumors in which nonpathogenic, opine-utilizing agrobacteria often prevail (4, 14). To account for the frequent occurrence of nonpathogenic agrobacteria, it is proposed here that some plant infections with A. tumefaciens result in the modification of the bacterial genome. Conceivably, nonpathogenic mutants produced by A. tumefaciens in association with the host plant could be altered either in the vir or T region of the Ti plasmid or in the chromosome. Chromosomal genes that are required for efficient plant transformation include chvE, which encodes a protein that potentiates the effect of plant phenolic compounds on vir gene induction (11).Our recent observatio...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%