Limits...
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.

Show MeSH

Related in: MedlinePlus

Suppression of A. tumefaciens tumorigenesis in oncogene-silenced walnut microshoots. Walnut microshoots were inoculated with virulent A. tumefaciens strain 20W-5A. A) Inoculation sites of untransformed microshoots formed large, undifferentiated tumors five weeks post-inoculation. B) Oncogene-silenced line 33-3-1 exhibited no tumor development. C) Small fragments of callus from A. tumefaciens inoculation sites cultured on hormone-free plant growth medium for five weeks: untransformed callus (upper) and callus derived from transgenic line 33-3-1 (lower).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3852553&req=5

Figure 5: Suppression of A. tumefaciens tumorigenesis in oncogene-silenced walnut microshoots. Walnut microshoots were inoculated with virulent A. tumefaciens strain 20W-5A. A) Inoculation sites of untransformed microshoots formed large, undifferentiated tumors five weeks post-inoculation. B) Oncogene-silenced line 33-3-1 exhibited no tumor development. C) Small fragments of callus from A. tumefaciens inoculation sites cultured on hormone-free plant growth medium for five weeks: untransformed callus (upper) and callus derived from transgenic line 33-3-1 (lower).

Mentions: Oncogene (iaaM and ipt) silencing was screened phenotypically following in vitro application of A. tumefaciens to sixteen microshoots generated from transformed line 33-3-1 or untransformed controls. The amount of undifferentiated callus forming on these tissues after inoculation with virulent A. tumefaciens strain 20W-5A was assayed five weeks post-inoculation (Figure 5A and B). Untransformed controls formed galls on 14 of 16 microshoots inoculated whereas no galls were formed on shoots of the transformed line 33. Control lines developed green callus at inoculation sites after five weeks. To confirm that this callus was crown gall tissue, small pieces were cultured further on hormone-free DKW medium. While most walnut tissues (including wound callus) will not proliferate on this medium, crown gall tissue is capable of rapid hormone-independent growth. Callus excised from controls displayed vigorous hormone-independent expansion while callus excised from line 33 displayed only minimal growth (Figure 5C). These results indicate crown gall initiation and proliferation is suppressed in transgenic line 33.


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)

Suppression of A. tumefaciens tumorigenesis in oncogene-silenced walnut microshoots. Walnut microshoots were inoculated with virulent A. tumefaciens strain 20W-5A. A) Inoculation sites of untransformed microshoots formed large, undifferentiated tumors five weeks post-inoculation. B) Oncogene-silenced line 33-3-1 exhibited no tumor development. C) Small fragments of callus from A. tumefaciens inoculation sites cultured on hormone-free plant growth medium for five weeks: untransformed callus (upper) and callus derived from transgenic line 33-3-1 (lower).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Suppression of A. tumefaciens tumorigenesis in oncogene-silenced walnut microshoots. Walnut microshoots were inoculated with virulent A. tumefaciens strain 20W-5A. A) Inoculation sites of untransformed microshoots formed large, undifferentiated tumors five weeks post-inoculation. B) Oncogene-silenced line 33-3-1 exhibited no tumor development. C) Small fragments of callus from A. tumefaciens inoculation sites cultured on hormone-free plant growth medium for five weeks: untransformed callus (upper) and callus derived from transgenic line 33-3-1 (lower).
Mentions: Oncogene (iaaM and ipt) silencing was screened phenotypically following in vitro application of A. tumefaciens to sixteen microshoots generated from transformed line 33-3-1 or untransformed controls. The amount of undifferentiated callus forming on these tissues after inoculation with virulent A. tumefaciens strain 20W-5A was assayed five weeks post-inoculation (Figure 5A and B). Untransformed controls formed galls on 14 of 16 microshoots inoculated whereas no galls were formed on shoots of the transformed line 33. Control lines developed green callus at inoculation sites after five weeks. To confirm that this callus was crown gall tissue, small pieces were cultured further on hormone-free DKW medium. While most walnut tissues (including wound callus) will not proliferate on this medium, crown gall tissue is capable of rapid hormone-independent growth. Callus excised from controls displayed vigorous hormone-independent expansion while callus excised from line 33 displayed only minimal growth (Figure 5C). These results indicate crown gall initiation and proliferation is suppressed in transgenic line 33.

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