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Stacking resistance to crown gall and nematodes in walnut rootstocks.

Walawage SL, Britton MT, Leslie CA, Uratsu SL, Li Y, Dandekar AM - BMC Genomics (2013)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Plant Sciences, University of California Davis, One Shields Avenue, Davis, CA, 95616, USA. amdandekar@ucdavis.edu.

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.

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Inhibition of root lesion nematodes in roots of co-transformed somatic embryos of genotype J1. Nematode infestation is expressed as the number of nematodes recovered from cultures initiated using 100 nematodes per rooted embryo. Nematodes were recovered per root for each transgenic line after two months of in vitro co-culture in the dark. Bars represent mean of three replicates (Error bars=S.D). Significant differences from controls are denoted with *. Two lines are significantly different whenever they have no letters in common. Comparisons are considered significant whenever p<0.05.
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Figure 2: Inhibition of root lesion nematodes in roots of co-transformed somatic embryos of genotype J1. Nematode infestation is expressed as the number of nematodes recovered from cultures initiated using 100 nematodes per rooted embryo. Nematodes were recovered per root for each transgenic line after two months of in vitro co-culture in the dark. Bars represent mean of three replicates (Error bars=S.D). Significant differences from controls are denoted with *. Two lines are significantly different whenever they have no letters in common. Comparisons are considered significant whenever p<0.05.

Mentions: A rapid screening method was used to test for nematode resistance in both co-transformed and single-vector transformed transgenic lines. The nematode population supported by each transgenic line was compared to untransformed (J1 and RR4) and transgenic (Pv194-8) controls. Results of these trials are shown in the figures below (FiguresĀ 1, 2, 3 and 4).


Stacking resistance to crown gall and nematodes in walnut rootstocks.

Walawage SL, Britton MT, Leslie CA, Uratsu SL, Li Y, Dandekar AM - BMC Genomics (2013)

Inhibition of root lesion nematodes in roots of co-transformed somatic embryos of genotype J1. Nematode infestation is expressed as the number of nematodes recovered from cultures initiated using 100 nematodes per rooted embryo. Nematodes were recovered per root for each transgenic line after two months of in vitro co-culture in the dark. Bars represent mean of three replicates (Error bars=S.D). Significant differences from controls are denoted with *. Two lines are significantly different whenever they have no letters in common. Comparisons are considered significant whenever p<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852553&req=5

Figure 2: Inhibition of root lesion nematodes in roots of co-transformed somatic embryos of genotype J1. Nematode infestation is expressed as the number of nematodes recovered from cultures initiated using 100 nematodes per rooted embryo. Nematodes were recovered per root for each transgenic line after two months of in vitro co-culture in the dark. Bars represent mean of three replicates (Error bars=S.D). Significant differences from controls are denoted with *. Two lines are significantly different whenever they have no letters in common. Comparisons are considered significant whenever p<0.05.
Mentions: A rapid screening method was used to test for nematode resistance in both co-transformed and single-vector transformed transgenic lines. The nematode population supported by each transgenic line was compared to untransformed (J1 and RR4) and transgenic (Pv194-8) controls. Results of these trials are shown in the figures below (FiguresĀ 1, 2, 3 and 4).

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Plant Sciences, University of California Davis, One Shields Avenue, Davis, CA, 95616, USA. amdandekar@ucdavis.edu.

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.

Show MeSH
Related in: MedlinePlus