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.     

Polymyxin resistance in Agrobacterium tumefaciens and its effect on crown gall tumor induction.

Polymyxin-resistant (PBLr) mutants of Agrobacterium tumefaciens A6, B6, and B6M were isolated from polymyxin-sensitive (PBLs) parent strains in a defined medium containing 600 microgram of polymyxin B sulfate per millilitre. The weight and number of tumors induced by PBLr mutants on a variety of host plants such as carrot, potato, and pinto bean were 45--75% less than those induced by PBLs wild types. The crude cell envelopes (CCE) prepared from both PBLs and PBLr bacteria were inhibitory for tumor initiation when they were applied before or during the inoculation of viable tumorigenic bacteria, but not when they were applied 30 min after the inoculation of infectious bacteria. The potency to inhibit the tumor initiation by the CCE prepared from PBLs cells was approximately 50% higher than that by the equal amount of the CCE prepared from PBLr cells. The concentration of CCE preparations required to reduce tumor induction 50% in carrot and pinto bean was determined to be 2.6 mg/mL and 4.0--6.2 mg/mL for the CCE derived from PBLs and PBLr cells, respectively. These data suggest that the envelope structure or composition of PBLs and PBLr cells is distinct, and that the acquisition of resistance to polymyxin by agrobacteria modifies envelope structure or components which are essential for tumor initiation.     

Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism.

Mutants of Agrobacterium tumefaciens which affect virulence or the ability to catabolize octopine were isolated after Tn5-induced mutagenesis. Of 8,900 colonies tested, 7 mutants with Tn5 insertions in a specific region of other Ti plasmid unable to catabolize octopine were isolated. Thirty-seven mutants affected in tumorigenesis resulted from insertions in the Ti plasmid and the Agrobacterium chromosome. Of these mutations, 12 were chromosomal and 25 mapped on the plasmid. Twenty-three mapped within a 20-megadalton region, which is distinct from the Ti plasmid sequences found stably integrated into the plant cell genome T-deoxyribonucleic acid). Included in these were mutants that were either a virulent or produced tumors with unusual morphologies. Three mutants contained insertions in the T-deoxyribonucleic acid. These three mutants incited tumors which synthesized octopine but had an altered morphology due to either extensive proliferation of shoots or roots from the tumor callus. Three additional mutants not caused by Tn5 contained mutations in the Ti plasmid.     

Biochemical and genetic characterisation of Agrobacterium tumefaciens the causal agent of walnut crown gall disease in Iran

In 2009–2010, crown tumours were collected from walnut (Juglans regia L.) trees in northern Iran. Gram-negative, rod shaped and aerobic bacteria with circular, convex and white-coloured colonies on potato dextrose agar plus CaCO3 medium were isolated from galls. In pathogenicity tests, tomato seedlings were inoculated with all strains and tumours started to appear three weeks after inoculation. Strains yielded a 224?bp amplicon from the virD2 gene in PCR. When the 16S rRNA gene sequence of strains was compared by BLASTn with nucleotide sequences from GenBank, it showed 99.6% identity with the 16S rRNA sequence of Agrobacterium tumefaciens ATCC 33970. Based on phenotypic and genotypic properties, the bacterium that causes crown gall of walnut trees was identified as A. tumefaciens.     

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.     

Salicylic acid, active oxygen species and systemic acquired resistance in plants

Infection of plants, particularly by a necrotizing pathogen, usually induces a long-lasting, broad-based, systemic resistance to secondary pathogen attack. Many studies implicate salicylic acid as an essential signal in the development of such systemic acquired resistance in several plant species. Salicylic acid appears to mediate plant defence by binding to and inhibiting catalase, thus increasing the concentration of H(2)O(2) and other active oxygen species. Active oxygen species may then act as second messengers that induce plant defence gene expression, analogous to their activation of gene expression in mammalian cells.     

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.     

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.     

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.     

D-glucaric acid and galactaric acid catabolism by Agrobacterium tumefaciens

Cell-free extract (crude extract) of Agrobacterium tumefaciens grown on d-glucuronate or d-glucarate converts d-glucarate and galactarate to a mixture of 2-keto-3-deoxy- and 4-deoxy-5-keto-d-glucarate. These compounds are then converted by partially purified crude extract to an intermediate tentatively identified as 2,5-diketoadipate. The same enzyme preparation further decarboxylates this intermediate to alpha-ketoglutarate semialdehyde, which is subsequently oxidized in a nicotinamide adenine dinucleotide-dependent reaction to alpha-ketoglutaric acid. Since A. tumefaciens converts d-glucuronic acid to d-glucarate, a pathway from d-glucuronate to alpha-ketoglutarate in A. tumefaciens was determined.