Biological control of crown gall, Agrobacterium tumefaciens

Biological control of crown gall caused by Agrobacteriurn turnefaciens (Smith & Townsend) Conn, pioneered by Dr A. Kerr in South Australia, is effected through the establishment of a high population of the related non-pathogen A. radiobacter (Beijerinck & van Delden) Conn, strain 84 in the rhizosphere of susceptible plants. Strain 84 produces a bacteriocin to which many strains of the pathogen in Australasia, North America and Britain are sensitive. The disease is present in Britain on a variety of hosts including cherry. At East Malling cherry leaf scars, invaluable as an avenue of infection for bacterial canker infectivity titrations, have been used successfully in crown gall studies. Live cells of strain 84, but neither an avirulent strain of the pathogen nor a soil bacterium highly antagonistic to A. tumefaciens in vitro, inhibited gall formation in cherry leaf scars. Heat-killed cells had no effect. In a field experiment at East Malling hardwood cuttings of the new rootstock Colt have been dipped in strain 84 and total inhibition of crown gall is expected to ensue. The results of other experiments where the disease is already established on cherry-rootstock layer-beds and in blackberry plantations are less predictable. In time we hope to solve this problem. Only time will show whether this method of biological control is long lasting or will eventually break down.     

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

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.     

Methylation of the T-DNA in Agrobacterium tumefaciens and in several crown gall tumors.

Methylation of the T-DNA in Agrobacterium tumefaciens and in four octopine-type (A6S/2, E9, 15955/1, 15955/01) and one nopaline-type (HT37#15) crown gall tumors was investigated using the isoschizomeric restriction endonucleases Msp I and Hpa II. T-DNA in the octopine-type Ti-plasmid pTiB6(806) was not methylated at the sequence 5'CCGG3' in Agrobacterium. With two possible exceptions, neither was the T-DNA of the nopaline-type Ti-plasmid pTiT37 methylated in the bacterium. In all tumor lines investigated, at least one copy of the T-DNA was not methylated. DNA methylation was not detected in the lines A6S/2, 15955/1, HT37#15, and the TL region of E9. DNA methylation of some copies of TR in the E9 tumor line, and possibly in the 15955/01 line, was detected. The methylation of some copies of TR in the E9 line may indicate that not all copies of TR are transcribed in this tumor.     

Highly tumorigenic Agrobacterium tumefaciens strain from crown gall tumors of chrysanthemum

A wild-type Agrobacterium tumefaciens strain CNI5 isolated from crown gall of chrysanthemum (Dendranthema grandiflora Tzvelev) was characterized. Strain CNI5 was classified into biovar 1, based on physiological and biochemical characteristics, and was resistant to ampicillin. Strain CNI5 induced tumors at a higher frequency and on a larger area of explants in most tested plant species, especially in chrysanthemum cultivars, than the octopine-type strain C58C1cmr (pTiB6S3). Agropine and mannopine were detected in tumors induced by strain CNI5 and were specifically catabolized by this strain. Strain CNI5 harbored five plasmids including one plasmid that shared sequence similarity to TL-DNA of the octopine-type Ti plasmid and four cryptic plasmids.     

Salicylic acid and systemic acquired resistance play a role in attenuating crown gall disease caused by Agrobacterium tumefaciens

We investigated the effects of salicylic acid (SA) and systemic acquired resistance (SAR) on crown gall disease caused by Agrobacterium tumefaciens. Nicotiana benthamiana plants treated with SA showed decreased susceptibility to Agrobacterium infection. Exogenous application of SA to Agrobacterium cultures decreased its growth, virulence, and attachment to plant cells. Using Agrobacterium whole-genome microarrays, we characterized the direct effects of SA on bacterial gene expression and showed that SA inhibits induction of virulence (vir) genes and the repABC operon, and differentially regulates the expression of many other sets of genes. Using virus-induced gene silencing, we further demonstrate that plant genes involved in SA biosynthesis and signaling are important determinants for Agrobacterium infectivity on plants. Silencing of ICS (isochorismate synthase), NPR1 (nonexpresser of pathogenesis-related gene 1), and SABP2 (SA-binding protein 2) in N. benthamiana enhanced Agrobacterium infection. Moreover, plants treated with benzo-(1,2,3)-thiadiazole-7-carbothioic acid, a potent inducer of SAR, showed reduced disease symptoms. Our data suggest that SA and SAR both play a major role in retarding Agrobacterium infectivity.     

Time required for tumor induction by Agrobacterium tumefaciens

Cellulose-minus mutants of Agrobacterium tumefaciens retain virulence but can be removed from wound sites by washing with water. Washing of Bryophyllum diagremontiana leaves inoculated with a cellulose-minus mutant was used to determine the minimum time the bacteria must be present for tumor induction. This time was 4 to 8 h.     

Silencing Agrobacterium oncogenes in transgenic grapevine results in strain-specific crown gall resistance

Key message

Grapevine rootstock transformed with an Agrobacterium oncogene-silencing transgene was resistant to certain Agrobacterium strains but sensitive to others. Thus, genetic diversity of Agrobacterium oncogenes may limit engineering crown gall resistance.

Abstract

Crown gall disease of grapevine induced by Agrobacterium vitis or Agrobacterium tumefaciens causes serious economic losses in viticulture. To establish crown gall-resistant lines, somatic proembryos of Vitis berlandieri × V. rupestris cv. ‘Richter 110’ rootstock were transformed with an oncogene-silencing transgene based on iaaM and ipt oncogene sequences from octopine-type, tumor-inducing (Ti) plasmid pTiA6. Twenty-one transgenic lines were selected, and their transgenic nature was confirmed by polymerase chain reaction (PCR). These lines were inoculated with two A. tumefaciens and three A. vitis strains. Eight lines showed resistance to octopine-type A. tumefaciens A348. Resistance correlated with the expression of the silencing genes. However, oncogene silencing was mostly sequence specific because these lines did not abolish tumorigenesis by A. vitis strains or nopaline-type A. tumefaciens C58.     

Studies on superoxide dismutase activities in virulent and avirulent strains of Agrobacterium tumefaciens and also in normal and crown gall tumor cells of Bryophyllum calycinum

Superoxide dismutase activity in virulent strains of Agrobacterium tumefaciens was found to be higher than that in avirulent strains. Polyacrylamide gel electrophoresis revealed two isoenzymes in both these strains. These isoenzymes are suggested to be iron and manganese containing superoxide dismutases. Crown gall tumor cells of the plant Bryophyllum calycinum were found to have higher superoxide dismutase activity than the normal plant cells. Polyacrylamide gel electrophoresis revealed two isoenzymes in both normal and crown gall tumor cells. Advantages of the higher superoxide dismutase activities in respect of the survival of virulent strains of A. tumefaciens and crown gall tumor growth have been discussed.     

Correlation between binding of Agrobacterium tumefaciens by root cap cells and susceptibility of plants to crown gall

We compared the binding of Agrobacterium tumefaciens by freshly isolated root cap cells with susceptibility of plants to crown gall tumorigenesis. A high binding reaction was strongly correlated with susceptibility to tumorigenesis in a survey of the binding of strain B6 to cells from 48 species in 17 families. In reciprocal experiments with nine virulent A. tumefaciens strains, tumors developed in plant-bacteria combinations that gave a high binding response in the root cap cell assay. Binding was quantified by direct measurement of the number of bacteria bound to the periphery of individual cells. Root cap cells from six susceptible species bound significantly more bacteria than did cells from five resistant species.     

Polyamines and crown gall tumor growth

The effect of the polyamine spermidine on the growth of crown gall tumors was determined using the potato disc bioassay. Addition of lmM spermidine resulted in a 30–50% increase in tumor growth. The spermidine effect was found to be biphasic, with lmM being optimal. Closely related polyamines including spermine, as well as other nitrogen containing compounds such as arginine and alanine, failed to promote tumor growth or inhibited the growth of these tumors. Endogenous levels of spermidine in crown gall tumor tissue were consistently greater than those of corresponding normal potato tissue. Rapidly dividing normal potato tissue derived from buds also contained elevated spermidine levels.     

Inter-organ defense networking: Leaf whitefly sucking elicits plant immunity to crown gall disease caused by Agrobacterium tumefaciens

Plants have elaborate defensive machinery to protect against numerous pathogens and insects. Plant hormones function as modulators of defensive mechanisms to maintain plant resistance to natural enemies. Our recent study suggests that salicylic acid (SA) is the primary phytohormone regulating plant responses to Agrobacterium tumefaciens infection. Tobacco (Nicotiana benthamiana Domin.) immune responses against Agrobacterium-mediated crown gall disease were activated by exposure to the sucking insect whitefly, which stimulated SA biosynthesis in aerial tissues; in turn, SA synthesized in aboveground tissues systemically modulated SA secretion in root tissues. Further investigation revealed that endogenous SA biosynthesis negatively modulated Agrobacterium-mediated plant genetic transformation. Our study provides novel evidence that activation of the SA-signaling pathway mediated by a sucking insect infestation has a pivotal role in subsequently attenuating Agrobacterium infection. These results demonstrate new insights into interspecies cross-talking among insects, plants, and soil bacteria.     

Effect of gibberellic Acid on crown gall tumor induction in aging primary pinto bean leaves

Gibberellic acid was tested for its effect on tumor induction by Agrobacterium tumefaciens in primary pinto bean (Phaseolus vulgaris) leaves in various stages of development. The hormone was found to promote tumor induction in partially aged leaves but did not effect tumor induction in very young leaves or in fully matured leaves. It is suggested that the natural loss of susceptibility to tumor induction in maturing pinto bean leaves is associated with a concomitant loss of endogenous gibberellins and/or a sensitivity to gibberellins.     

Intracellular accumulation of mannopine, an opine produced by crown gall tumors, transiently inhibits growth of Agrobacterium tumefaciens

pYDH208, a cosmid clone from the octopine-mannityl opine-type tumor-inducing (Ti) plasmid pTi15955 confers utilization of mannopine (MOP) and agropine (AGR) on Agrobacterium tumefaciens strain NT1. NT1 harboring pYDH208 with an insertion mutation in mocC, which codes for MOP oxidoreductase, not only fails to utilize MOP as a sole carbon source, but also was inhibited in its growth by MOP and AGR. In contrast, the growth of mutants with insertions in other tested moc genes was not inhibited by either opine. Growth of strains NT1 or UIA5, a derivative of C58 that lacks pAtC58, was not inhibited by MOP, but growth of NT1 or UIA5 harboring pRE10, which codes for the MOP transport system, was inhibited by the opine. When a clone expressing mocC was introduced, the growth of strain NT1(pRE10) was not inhibited by MOP, although UIA5(pRE10) was still weakly inhibited. In strain NT1(pRE10, mocC), santhopine (SOP), produced by the oxidation of MOP by MocC, was further degraded by functions encoded by pAtC58. These results suggest that MOP and, to a lesser extent, SOP are inhibitory when accumulated intracellularly. The growth of NT1(pRE10), as measured by turbidity and viable cell counts, ceased upon the addition of MOP but restarted in a few hours. Regrowth was partly the result of the outgrowth of spontaneous MOP-resistant mutants and partly the adaptation of cells to MOP in the medium. Chrysopine, isochrysopine, and analogs of MOP in which the glutamine residue is substituted with other amino acids were barely taken up by NT1(pRE10) and were not inhibitory to growth of the strain. Sugar analogs of MOP were inhibitory, and those containing sugars in the D form were more inhibitory than those containing sugars in the L form. MOP analogs containing hexose sugars were more inhibitory than those containing sugars with three, four, or five carbon atoms. Mutants of NT1(pRE10) that are resistant to MOP arose in the zone of growth inhibition. Genetic and physiological analyses indicate that the mutations are located on pRE10 and abolish uptake of the opine.