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Stacking resistance to crown gall and nematodes in walnut rootstocks.

ABSTRACT: BACKGROUND: Crown gall (CG) (Agrobacterium tumefaciens) and the root lesion nematodes (RLNs) (Pratylenchus vulnus) are major challenges faced by the California walnut industry, reducing productivity and increasing the cost of establishing and maintaining orchards. Current nematode control strategies include nematicides, crop rotation, and tolerant cultivars, but these methods have limits. Developing genetic resistance through novel approaches like RNA interference (RNAi) can address these problems. RNAi-mediated silencing of CG disease in walnut (Juglans regia L.) has been achieved previously. We sought to place both CG and nematode resistance into a single walnut rootstock genotype using co-transformation to stack the resistance genes. A. tumefaciens, carrying self-complimentary iaaM and ipt transgenes, and Agrobacterium rhizogenes, carrying a self-complimentary Pv010 gene from P. vulnus, were used as co-transformation vectors. RolABC genes were introduced by the resident T-DNA in the A. rhizogenes Ri-plasmid used as a vector for plant transformation. Pv010 and Pv194 (transgenic control) genes were also transferred separately using A. tumefaciens. To test for resistance, transformed walnut roots were challenged with P. vulnus and microshoots were challenged with a virulent strain of A. tumefaciens. RESULTS: Combining the two bacterial strains at a 1:1 rather than 1:3 ratio increased the co-transformation efficiency. Although complete immunity to nematode infection was not observed, transgenic lines yielded up to 79% fewer nematodes per root following in vitro co-culture than untransformed controls. Transgenic line 33-3-1 exhibited complete crown gall control and 32% fewer nematodes. The transgenic plants had thicker, longer roots than untransformed controls possibly due to insertion of rolABC genes. When the Pv010 gene was present in roots with or without rolABC genes there was partial or complete control of RLNs. Transformation using only one vector showed 100% control in some lines. CONCLUSIONS: CG and nematode resistance gene stacking controlled CG and RLNs simultaneously in walnuts. Silencing genes encoding iaaM, ipt, and Pv010 decrease CG formation and RLNs populations in walnut. Beneficial plant genotype and phenotype changes are caused by co-transformation using A. tumefaciens and A. rhizogenes strains. Viable resistance against root lesion nematodes in walnut plants may be accomplished in the future using this gene stacking technology.

SUBMITTER: Walawage SL

PROVIDER: S-EPMC3852553 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Stacking resistance to crown gall and nematodes in walnut rootstocks.

Walawage Sriema L SL Britton Monica T MT Leslie Charles A CA Uratsu Sandra L SL Li YingYue Y Dandekar Abhaya M AM

BMC genomics 20131001

<h4>Background</h4>Crown gall (CG) (Agrobacterium tumefaciens) and the root lesion nematodes (RLNs) (Pratylenchus vulnus) are major challenges faced by the California walnut industry, reducing productivity and increasing the cost of establishing and maintaining orchards. Current nematode control strategies include nematicides, crop rotation, and tolerant cultivars, but these methods have limits. Developing genetic resistance through novel approaches like RNA interference (RNAi) can address these ...[more]

PMID: 24083348

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Project description:BackgroundCrown gall disease, caused by the pathogenic bacterium Agrobacterium tumefaciens, is responsible for extensive economic losses in orchards. Cherry rootstock 'CDR-1' (Prunus mahaleb) shows high resistance but the mechanism remains unclear. Here, we examined the morphology of pathogen-infected root neck surface, determined the activity of 10 defense-related enzymes and the content of salicylic acid (SA) and jasmonic acid (JA), and also applied transcriptome analysis, transient expression and transgenic verification to explore the crown gall resistance genes in 'CDR-1' plants.ResultsIn our study, peroxidase increased in the first 10 days, while phenylalanine ammonialyase and lipoxygenase increased in the first 15 days post-infection. Four key enzymes in the AsA-GSH cycle also responded, to a certain extent; although JA content increased significantly after the treatment, the SA content did not. In a follow-up transcriptome analysis, the differentially expressed genes Pm4CL2, PmCYP450, PmHCT1, PmHCT2, and PmCAD were up-regulated. Based on the above results, we focused on the lignin biosynthetic pathway, and further measured lignin content, and found it increased significantly. The Pm4CL2 gene was used to conduct transient expression and transgenic experiments to verify its function in crown gall disease resistance. It showed the relative expression of the treatment group was almost 14-fold that of the control group at 12 h post-treatment. After the infection treatment, clear signs of resistance were found in the transgenic lines; this indicated that under the higher expression level and earlier activation of Pm4CL2, plant resistance was enhanced.ConclusionsThe crown gall resistance of 'CDR-1' is likely related to the lignin biosynthetic pathway, in which Pm4CL2 functions crucially during the plant defense response to the pathogen A. tumefaciens. The results thus offer novel insights into the defense responses and resistance mechanism of cherry rootstock 'CDR-1' against crown gall disease.

Project description:Crown gall disease caused by Agrobacterium tumefaciens severely impacts the production of peach and other fruit trees. Several peach cultivars are partially resistant to A. tumefaciens, but little is known about the roles of endophytic microbiota in disease resistance. In the present study, the endophytic bacterial communities of resistant and susceptible peach cultivars "Honggengansutao" and "Okinawa" were analyzed using universal 16S rRNA gene amplicon sequencing in parallel with the cultivation and characterization of bacterial isolates. A total of 1,357,088 high-quality sequences representing 3,160 distinct operational taxonomic units (OTUs; Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes) and 1,200 isolates of 20 genera and 305 distinct ribotypes were collected from peach roots and twigs. It was found that factors including plant developmental stage, cultivar, and A. tumefaciens invasion strongly influenced the peach endophytic communities. The community diversity of endophytic bacteria and the abundance of culturable bacteria were both higher in the roots of the resistant cultivar, particularly after inoculation. Strikingly, the pathogen antagonists Streptomyces and Pseudomonas in roots and Rhizobium in twigs were most frequently detected in resistant plants. Our results suggest that the higher abundance and diversity of endophytic bacteria and increased proportions of antagonistic bacteria might contribute to the natural defense of the resistant cultivar against A. tumefaciens This work reveals the relationships between endophytic bacteria and disease resistance in peach plants and provides important information for microbiome-based biocontrol of crown gall disease in fruit trees.IMPORTANCEAgrobacterium tumefaciens as the causal agent of peach crown gall disease can be controlled by planting resistant cultivars. This study profiles the endophytic bacteria in susceptible and resistant peach cultivars, advancing our understanding of the relationships between endophytic bacterial communities and peach crown gall disease, with potential implications for other complex microbiome-plant-pathogen interactions. The resistant cultivar may defend itself by increasing the diversity and abundance of beneficial endophytic bacteria. The antagonists identified among the genera Streptomyces, Pseudomonas, and Rhizobium may have application potential for biocontrol of crown gall disease in fruit trees.

Project description: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.

Dataset Information

Stacking resistance to crown gall and nematodes in walnut rootstocks.

Publications

Stacking resistance to crown gall and nematodes in walnut rootstocks.

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