Crown Gall Development Research Articles

Crown gall development on cannabis (Cannabis sativa L., marijuana) plants caused by Agrobacterium tumefaciens species-complex

Crown gall, caused by Agrobacterium tumefaciens (syn. Rhizobium radiobacter), is characterized by gall formation on crowns, stems or roots on many plant species. We describe the occurrence of this disease for the first time on cannabis (Cannabis sativa L. marijuana) plants in two licenced production facilities in British Columbia. Galls were observed on crowns, stems and roots of several genotypes, including ‘White Rhino’, ‘Pink Kush’ and ‘Sour Kush’. The affected plants displayed no other visible symptoms. The incidence of crown gall was extremely low, estimated at 0.01% of total plants. Isolations made from root and stem galls on MacConkey medium (selective for Gram -ve bacteria) and on D1 medium (selective for Agrobacterium spp.) yielded a range of bacterial species, which did not include Agrobacterium sp. However, the presence of Agrobacterium in gall tissues was confirmed following PCR amplification with primers for the indole-acetic acid (iaa) H gene on the T-DNA region of Agrobacterium spp. and showed > 99% similarity to A. tumefaciens. Artificial inoculations were conducted using an A. tumefaciens strain from ATCC (Rhizobium radiobacter, strain designation TT134 [6-1-2, CIP 104 336], cat. 15955) which resulted in gall formation on five cannabis genotypes tested. These galls contained the iaaH gene as determined by PCR and the pathogen was successfully re-isolated on MacConkey agar medium at 4 and 10 weeks post-inoculation. Our findings confirm the occurrence of A. tumefaciens on naturally infected and artificially infected cannabis plants grown under greenhouse conditions and demonstrate reproducible gall symptoms on several cannabis genotypes following A. tumefaciens inoculation.

Transcriptome analysis of crown gall in radish (Raphanus sativus L.) inbred lines

AbstractStudying aberrations of normal developmental processes like plant tumour initiation provides a great opportunity for understanding of systemic control of plant cells differentiation. Most well‐studied example of plant tumour is the crown gall caused by Agrobacterium tumefaciens; much less studied are spontaneous tumours developing in plants with certain genotype, regardless of agrobacterial infection. In the present study, we used RNA‐seq to analyse the changes in gene expression at the early stages of development of A. tumefaciens‐induced tumours in radish seedlings. In our studies we used two closely related radish inbred lines contrasting in their ability to form spontaneous tumours. RNA‐seq and qPCR analysis showed significant changes in the expression levels for numerous genes involved in different metabolic and developmental processes, and Gene Ontology analysis indicated many biological processes probably involved in the crown gall development in radish. For example, a unique result of our research is upregulation of genes related to photomorphogenesis and downregulation of senescence‐associated genes in the young crown gall tumours. Differences in gene expression between the analysed radish lines included upregulation of pathways related to chromatin remodelling and protein targeting to chloroplast in the tumorous line along with downregulation of pathways of chaperone‐mediated complex assembly and anthocyanin biosynthesis.

Effect of potential biocontrol agents selected among grapevine endophytes and commercial products on crown gall disease

The current strategies for the control of Agrobacterium vitis crown gall in grape are generally unsuccessful once the pathogen has established in vineyards. Experimental trials were conducted to evaluate the effectiveness of treatments based on non-pathogenic endophytes isolated from asymptomatic grapevines growing in vineyards with high incidence of crown gall and on microorganisms isolated from commercial products. Two-year in planta trials conducted on rootstocks treated with endophytic isolates showed the effectiveness of two bacterial endophytes, both in the genus Curtobacterium, and one fungal isolate in the genus Acremonium in reducing crown gall development. For the commercial biological control agents, Bacillus subtilis SR63 and Trichoderma asperellum T1 were the most effective strains against A. vitis, indicating commercial products could be reserves to draw upon to identify useful biocontrol agents. Based on the combination of data in this work, microorganisms, both endophytes and those formulated in commercial products, were identified that can potentially be exploited for the control of grapevine crown gall disease.

The Nonspecific Lipid Transfer Protein AtLtpI-4 Is Involved in Suberin Formation of Arabidopsis thaliana Crown Galls.

Nonspecific lipid transfer proteins reversibly bind different types of lipid molecules in a hydrophobic cavity. They facilitate phospholipid transfer between membranes in vitro, play a role in cuticle and possibly in suberin formation, and might be involved in plant pathogen defense signaling. This study focuses on the role of the lipid transfer protein AtLTPI-4 in crown gall development. Arabidopsis (Arabidopsis thaliana) crown gall tumors, which develop upon infection with the virulent Agrobacterium tumefaciens strain C58, highly expressed AtLTPI-4 Crown galls of the atltpI-4 loss-of-function mutant were much smaller compared with those of wild-type plants. The gene expression pattern and localization of the protein to the plasma membrane pointed to a function of AtLTPI-4 in cell wall suberization. Since Arabidopsis crown galls are covered by a suberin-containing periderm instead of a cuticle, we analyzed the suberin composition of crown galls and found a reduction in the amounts of long-chain fatty acids (C18:0) in the atltpI-4 mutant. To demonstrate the impact of AtLtpI-4 on extracellular lipid composition, we expressed the protein in Arabidopsis epidermis cells. This led to a significant increase in the very-long-chain fatty acids C24 and C26 in the cuticular wax fraction. Homology modeling and lipid-protein-overlay assays showed that AtLtpI-4 protein can bind these very-long-chain fatty acids. Thus, AtLtpI-4 protein may facilitate the transfer of long-chain as well as very-long-chain fatty acids into the apoplast, depending on the cell type in which it is expressed. In crown galls, which endogenously express AtLtpI-4, it is involved in suberin formation.

Biological control agent Agrobacterium vitis strain ARK-1 suppresses expression of the virD2 and virE2 genes in tumorigenic A. vitis

A nonpathogenic strain of Agrobacterium (=Rhizobium) vitis, ARK-1, limits the development of crown gall of grapevine (Vitis vinifera L.). Co-inoculation of grapevine shoots with ARK-1 and the tumorigenic (Ti) strain VAT03-9 at a 1:1 cell ratio resulted in significantly lower expression of the virulence genes virD2 and virE2 of VAT03-9 1 day after inoculation (dai) compared with expression levels when shoots were inoculated only with VAT03-9. In contrast, nonpathogenic A. vitis strain VAR06-30, which does not limit the development of crown gall of grapevine, co-inoculated with VAT03-9 did not affect expression levels of virD2 and virE2 under the same conditions. ARK-1 began to suppress the VAT03-9 population by seven dai, but no such effect was observed with VAR06-30 during the nine dai study period. Thus, the biological control activity of ARK-1 is likely based on the suppression of essential virulence genes.

Regulation of oncogene expression in T-DNA-transformed host plant cells.

Virulent Agrobacterium tumefaciens strains integrate their T-DNA into the plant genome where the encoded agrobacterial oncogenes are expressed and cause crown gall disease. Essential for crown gall development are IaaH (indole-3-acetamide hydrolase), IaaM (tryptophan monooxygenase) and Ipt (isopentenyl transferase), which encode enzymes for the biosynthesis of auxin (IaaH, IaaM) and cytokinin (Ipt). Although these oncogenes are well studied as the tumor-inducing principle, nothing is known about the regulation of oncogene expression in plant cells. Our studies show that the intergenic regions (IGRs) between the coding sequences (CDS) of the three oncogenes function as promoters in plant cells. These promoters possess a eukaryotic sequence organization and cis-regulatory elements for the binding of plant transcription factors. WRKY18, WRKY40, WRKY60 and ARF5 were identified as activators of the Ipt promoter whereas IaaH and IaaM is constitutively expressed and no transcription factor further activates their promoters. Consistent with these results, the wrky triple mutant plants in particular, develops smaller crown galls than wild-type and exhibits a reduced Ipt transcription, despite the presence of an intact ARF5 gene. WRKY40 and WRKY60 gene expression is induced by A. tumefaciens within a few hours whereas the ARF5 gene is transcribed later during crown gall development. The WRKY proteins interact with ARF5 in the plant nucleus, but only WRKY40 together with ARF5 synergistically boosts the activation of the Ipt promoter in an auxin-dependent manner. From our data, we propose that A. tumefaciens initially induces WRKY40 gene expression as a pathogen defense response of the host cell. The WRKY protein is recruited to induce Ipt expression, which initiates cytokinin-dependent host cell division. With increasing auxin levels triggered by ubiquitous expression of IaaH and IaaM, ARF5 is activated and interacts with WRKY40 to potentiate Ipt expression and balance cytokinin and auxin levels for further cell proliferation.

Plant responses to Agrobacterium tumefaciens and crown gall development

Agrobacterium tumefaciens causes crown gall disease on various plant species by introducing its T-DNA into the genome. Therefore, Agrobacterium has been extensively studied both as a pathogen and an important biotechnological tool. The infection process involves the transfer of T-DNA and virulence proteins into the plant cell. At that time the gene expression patterns of host plants differ depending on the Agrobacterium strain, plant species and cell-type used. Later on, integration of the T-DNA into the plant host genome, expression of the encoded oncogenes, and increase in phytohormone levels induce a fundamental reprogramming of the transformed cells. This results in their proliferation and finally formation of plant tumors. The process of reprogramming is accompanied by altered gene expression, morphology and metabolism. In addition to changes in the transcriptome and metabolome, further genome-wide (“omic”) approaches have recently deepened our understanding of the genetic and epigenetic basis of crown gall tumor formation. This review summarizes the current knowledge about plant responses in the course of tumor development. Special emphasis is placed on the connection between epigenetic, transcriptomic, metabolomic, and morphological changes in the developing tumor. These changes not only result in abnormally proliferating host cells with a heterotrophic and transport-dependent metabolism, but also cause differentiation and serve as mechanisms to balance pathogen defense and adapt to abiotic stress conditions, thereby allowing the coexistence of the crown gall and host plant.

Reduction in pathogen populations at grapevine wound sites is associated with the mechanism underlying the biological control of crown gall by rhizobium vitis strain ARK-1.

A nonpathogenic strain of Rhizobium (=Agrobacterium) vitis, ARK-1, limited the development of grapevine crown gall. A co-inoculation with ARK-1 and the tumorigenic strain VAT07-1 at a 1:1 cell ratio resulted in a higher population of ARK-1 than VAT07-1 in shoots without tumors, but a significantly lower population of ARK-1 than VAT07-1 in grapevine shoots with tumors. ARK-1 began to significantly suppress the VAT07-1 population 2 d after the inoculation. This result indicated that ARK-1 reduced the pathogen population at the wound site through biological control. Although ARK-1 produced a zone of inhibition against other tumorigenic Rhizobium spp. in in vitro assays, antibiosis depended on the culture medium. ARK-1 did not inhibit the growth of tumorigenic R. radiobacter strain AtC1 in the antibiosis assay, but suppressed the AtC1-induced formation of tumors on grapevine shoots, suggesting that antibiosis by ARK-1 may not be the main mechanism responsible for biological control.

Role of hormones in controlling vascular differentiation and the mechanism of lateral root initiation

The vascular system in plants is induced and controlled by streams of inductive hormonal signals. Auxin produced in young leaves is the primary controlling signal in vascular differentiation. Its polar and non-polar transport pathways and major controlling mechanisms are clarified. Ethylene produced in differentiating protoxylem vessels is the signal that triggers lateral root initiation, while tumor-induced ethylene is a limiting and controlling factor of crown gall development and its vascular differentiation. Gibberellin produced in mature leaves moves non-polarly and promotes elongation, regulates cambium activity and induces long fibers. Cytokinin from the root cap moves upward to promote cambial activity and stimulate shoot growth and branching, while strigolactone from the root inhibits branching. Furthermore, the role of the hormonal signals in controlling the type of differentiating vascular elements and gradients of conduit size and density, and how they regulate plant adaptation and have shaped wood evolution are elucidated.

DNA Methylation Mediated Control of Gene Expression Is Critical for Development of Crown Gall Tumors

Crown gall tumors develop after integration of the T-DNA of virulent Agrobacterium tumefaciens strains into the plant genome. Expression of the T-DNA–encoded oncogenes triggers proliferation and differentiation of transformed plant cells. Crown gall development is known to be accompanied by global changes in transcription, metabolite levels, and physiological processes. High levels of abscisic acid (ABA) in crown galls regulate expression of drought stress responsive genes and mediate drought stress acclimation, which is essential for wild-type-like tumor growth. An impact of epigenetic processes such as DNA methylation on crown gall development has been suggested; however, it has not yet been investigated comprehensively. In this study, the methylation pattern of Arabidopsis thaliana crown galls was analyzed on a genome-wide scale as well as at the single gene level. Bisulfite sequencing analysis revealed that the oncogenes Ipt, IaaH, and IaaM were unmethylated in crown galls. Nevertheless, the oncogenes were susceptible to siRNA–mediated methylation, which inhibited their expression and subsequently crown gall growth. Genome arrays, hybridized with methylated DNA obtained by immunoprecipitation, revealed a globally hypermethylated crown gall genome, while promoters were rather hypomethylated. Mutants with reduced non-CG methylation developed larger tumors than the wild-type controls, indicating that hypermethylation inhibits plant tumor growth. The differential methylation pattern of crown galls and the stem tissue from which they originate correlated with transcriptional changes. Genes known to be transcriptionally inhibited by ABA and methylated in crown galls became promoter methylated upon treatment of A. thaliana with ABA. This suggests that the high ABA levels in crown galls may mediate DNA methylation and regulate expression of genes involved in drought stress protection. In summary, our studies provide evidence that epigenetic processes regulate gene expression, physiological processes, and the development of crown gall tumors.

ACC deaminase activity in avirulentAgrobacterium tumefaciensD3

Some plant-growth-promoting bacteria encode the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which breaks down ACC, the direct precursor of ethylene biosynthesis in all higher plants, into ammonia and α-ketobutyrate and, as a result, reduces stress ethylene levels in plants caused by a wide range of biotic and abiotic stresses. It was previously shown that ACC deaminase can inhibit crown gall development induced by Agrobacterium tumefaciens and can partially protect plants from this disease. Agrobacterium tumefaciens D3 has been previously reported to contain a putative ACC deaminase structural gene (acdS) and a regulatory gene (acdR = lrpL). In the present study, it was found that A. tumefaciens D3 is an avirulent strain. ACC deaminase activity and its regulation were also characterized. Under gnotobiotic conditions, wild-type A. tumefaciens D3 was shown to be able to promote plant root elongation, while the acdS and lrpL double mutant strain A. tumefaciens D3-1 lost that ability. When co-inoculated with the virulent strain, A. tumefaciens C58, in wounded castor bean plants, both the wild-type A. tumefaciens D3 and the mutant A. tumefaciens D3-1 were found to be able to significantly inhibit crown gall development induced by A. tumefaciens C58.

ACC deaminase from plant growth-promoting bacteria affects crown gall development

In addition to the well-known roles of indoleacetic acid and cytokinin in crown gall formation, the plant hormone ethylene also plays an important role in this process. Many plant growth-promoting bacteria (PGPB) encode the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which can degrade ACC, the immediate precursor of ethylene in plants, to alpha-ketobutyrate and ammonia and thereby lower plant ethylene levels. To study the effect of ACC deaminase on crown gall development, an ACC deaminase gene from the PGPB Pseudomonas putida UW4 was introduced into Agrobacterium tumefaciens C58, so that the effect of ACC deaminase activity on tumour formation in tomato and castor bean plants could be assessed. Plants were also coinoculated with A. tumefaciens C58 and P. putida UW4 or P. putida UW4-acdS- (an ACC deaminase minus mutant strain). In both types of experiments, it was observed that the presence of ACC deaminase generally inhibited tumour development on both tomato and castor bean plants.

PHOSPHINOTHRICIN- AND CROWN GALL-RESISTANT TRANSGENIC PLANTS OF GRAPEVINE

ISHS XXVI International Horticultural Congress: Biotechnology in Horticultural Crop Improvement: Achievements, Opportunities and Limitations PHOSPHINOTHRICIN- AND CROWN GALL-RESISTANT TRANSGENIC PLANTS OF GRAPEVINE

Silencing crown gall disease in walnut ( Juglans regia L.)

Crown gall disease can severely limit the growth and productivity of many tree crops and ornamental plants. We have developed a novel crown gall disease resistance strategy that is based upon the silencing of two highly conserved Agrobacterium tumefaciens oncogenes which are required for crown gall development. The tryptophan monooxygenase ( iaaM) and isopentenyl transferase ( ipt) genes are horizontally transferred from A. tumefaciens to the plant cell, mediating the de novo auxin and cytokinin production that initiates tumorigenesis. By expressing inducers of post-transcriptional gene silencing homologous to iaaM and ipt in plant cells, the corresponding oncogene mRNA transcripts are degraded in planta, providing functional resistance to crown gall disease. This strategy is highly efficacious in English (Persian) walnut ( Juglans regia L.), as several transformed lines expressing self-complementary iaaM and ipt transgenes display a highly specific accumulation of small-interfering RNAs and a broad-spectrum suppression of tumorigenesis. Potential applications and limitations of this technology in the future development of crown gall-resistant fruit and nut crop are also discussed.

Control of crown gall in Swiss apple nurseries1

Experiments to control crown gall, caused by Agrobacterium tumefaciens, were carried out in an apple‐rootstock nursery. One‐year‐old apple rootstocks (M 9) were dipped in different concentrations of several copper compounds before planting. The compounds were used alone or in mixtures with clay or glue. Good control of crown gall was obtained with Copac E cuprammonium (ammoniacal cupric sulphate) and with copper oxychloride in clay slurries or mixed with glue. Although treatment with 2‐methoxyethylmercury chloride prevented the development of crown gall, phytotoxicity was severe.

Octopine Accumulation Early in Crown Gall Development is Progressive

A purification of octopine from crown gall tissue was developed to quantitate conversion of precursor [(3)H]arginine into [(3)H]octopine. Plant wound tissue which was sterile or infected with an avirulent strain of Agrobacterium tumefaciens did not accumulate detectable quantities of octopine, consistent with opine synthesis not being induced by wounding or infection. Octopine was only recovered from tissue infected with virulent tumor-inducing strains of A. tumefaciens. In every case tested, the morphological appearance of tumors preceded the accumulation of octopine by at least 1 week, and in some instances 3 weeks. Thus, what was necessary and sufficient for the expression of plant hormones (auxin and cytokinin) required for tumor growth was not sufficient for the accumulation of octopine. The possible nature of the temporal difference in the expression of hormone autotrophy and octopine synthesis is discussed.

Latent Infections and Seasonal Variability of Crown Gall Development in Seedlings of Three Prunus species

Latent Infections and Seasonal Variability of Crown Gall Development in Seedlings of Three Prunus species

The Role of Abscisic Acid in Sterile and Crown Gall Inoculated Wounds

Summary The significance of abscisic acid (ABA) in wound reaction and the early phase of crown gall development was studied by determining the ABA-content of treated internodes at specific intervals and by following the effect of exogenously applied ABA-solution on the growth of tumors and callus tissues. Still growing second distal internodes of Pisum sativum were used as the material. For the determination of endogenous ABA the internodes were wounded along their entire length either sterilely or with bacteria. Samples for gas-liquid chromatography were collected 0 and 20 hours as well as 2 and 4 days after the beginning of the experiment, and samples for a wheat coleoptile straight growth test after 1 and 4 days. The results indicate that wounding, both sterile and bacterial, caused changes in the inhibitor content of the tissue. Around sterile wounds the metabolism of ABA became disturbed at first. In four days' time, however, inhibitors accumulated again, and the amount of ABA may have risen above the normal level. Around inoculated wounds the catabolism of ABA was even more pronounced and the subsequent accumulation, if any, was slight. Qualitative differences in the inhibitor contents of the two kinds of tissue were also detected. It is suggested that the disturbance in the metabolism of ABA may be one of the factors releasing the DNA-synthesis in a mature tissue. The bacterial inoculation seems, amongst other things, to depress the subsequent accumulation of inhibitors. For the experiments with commercial ABA-solution one wedge-shaped wound was made per internode, and 2 μl of ABA (0.1–80 ppm) was applied both to the sterile and to the inoculated wounds either once (80 ppm) or four times (0.1, 2 and 20 ppm) at daily intervals. The size of the tumors and the amount of callus growth were determined after three weeks. None of the treatments affected the callus growth, nor did the one-dose application of inhibitor affect the tumor growth. With the four-dose treatments the response depended on the size of the plants: while in the bigger plants the tumor growth was significantly depressed, the tumors in the smaller plants thrived as well as or better than those in the untreated controls.

Crown Gall Development Stimulated by Wound Washing

AbstractA number of experiments on the effect of wound washing on the development of crown gall were made varying the following factors: Host species; bacterial strain; non‐washing versus washing for different periods; sterile versus inoculated wound; time of inoculation. Washing did not have a recognizable effect on the healing of sterile wounds. Neither did it seem to overcome incompatibility between host and bacterial strain. Washing promoted tumor growth significantly under the following conditions: Vicia faba inoculated with T37 20 hours after washing for 1 hour; Helianthus annuus inoculated with B6 immediately after washing for 2 hours.

Gravity Compensation and Crown Gall Development

CLINOSTATS provide a means for ground based studies of what happens when plants are subjected to null-gravity conditions1. The incubation period of leaf rust on gravity-compensated bean plants is shorter than on non-compensated controls2. Here we describe the influence of gravity compensation on crown gall development in the carrot (Daucus carota).