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

Abstract

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...

Agrobacterium -Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool

“…67,2003 AGROBACTERIUM-MEDIATED PLANT TRANSFORMATION 27 on May 9, 2018 by guest http://mmbr.asm.org/ have been revealed by various assays. Thus, rat1 (encoding an arabinogalactan protein) and rat3 (probably encoding a plant cell wall protein) are involved in bacterial attachment to roots (23). Other rat genes that may affect cell wall structure include a xylan synthase (rat4 [232]) and a ␤-expansin (A. Kaiser, A. Kopecki, Y. Zhu, and S. B. Gelvin, unpublished data).…”

“…However, the situation is likely to be more complex. Belanger et al (23) showed that individual virA genes may be particularly suited to function in certain genetic backgrounds, and Krishnamohan et al (183) recently demonstrated that Ti plasmids may have evolved to optimize specific combinations of virA, virG, and vir boxes. As noted above, the Ti-plasmid pTiBo542 in the C58 chromosomal background is hypervirulent on certain legume species, possibly because of the associated virG gene (41, 146, 159), but not in its native Bo542 chromosomal background (143).…”

Interplay of Darwinian and frequency-dependent selection in the host-associated microbial population

“…Напри мер, при инфицировании хозяина не отдельными клет ками, а их группами (проникновение цианобактерий в коралловидные корни саговников или в листовые же лезы Gunnera) этот процесс может быть описан с ис пользованием не стохастических, а более простых де терминистических подходов. При моделировании попу ляционной динамики агробактерий следует учитывать появление генетически измененных вариантов не толь ко в почве, но и в организме хозяина, где могут индуци роваться мутации [10] или перенос генов [7].…”

Variable efficiency of a Ti plasmid-encoded VirA protein in different agrobacterial hosts

“…Previous studies had shown that growth of strains D10B/87 and C58 was inhibited under conditions of vir gene induction, this inhibition favoring the selection of avirulent mutants which had become insensitive to the phenolic vir gene inducers (3,15). Because the transconjugant CB100 exhibited a defect in vir gene induction at acetosyringone concentrations of up to 100 M (see above), it appeared possible that this transconjugant would not produce avirulent mutants under FIG.…”