Stacking resistance to crown gall and nematodes in walnut rootstocks

Abstract

BackgroundCrown 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.ResultsCombining 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.ConclusionsCG 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.

RNA interference technology in crop protection against arthropod pests, pathogens and nematodes

“…This resulted in reduced infection by the nematode M. incognita , providing evidence that transgenic grapevines can be developed with resistance to nematodes via an RNAi approach. The use of RNAi to control parasitic root nematodes was also effective for other targets in Meloidogyne such as Phe‐met‐arg‐phe (FMRF)amide‐like peptide genes [encoding FMRF like peptides (flp)‐14 and flp‐18, which modulate nerve and muscle activity, respectively], but also for other genera such as Pratylenchus and Heterodera . All these examples provide a proof‐of‐concept for the potential to engineer resistance against a broad range of root‐knot nematodes in a variety of important agricultural plant species.…”

“…From a pest control perspective, ncRNAs trigger the RNAi mechanism, leading to gene silencing and blocking of the expression of essential target genes by destroying the corresponding messenger RNA (mRNA). 5,6 As a consequence of its highly specific mode of action compared with other pest control strategies such as conventional neurotoxic insecticides, RNAi technology comprises a suite of tools with a vast range of potential applications for genetic studies and agriculture, including protection of beneficial insects against viruses and parasites, 7,8 resistance management, 9 and pest control against insect species, 10-15 plant pathogens, [16][17][18][19][20][21][22][23] mites, [24][25][26] ticks, 27 and nematodes, 28,29 in a wide range of crops ( Fig. 1).…”

Walnut: past and future of genetic improvement

“…The iaaM and ipt genes of A. tumefaciens responsible for crown gall disease were silenced by expressing homologous inducers of post-transcriptional gene silencing (Escobar et al 2002). Co-transformation with A. tumefaciens (iaaM and ipt genes) and A. rhizogenes (Pv010 gene) was successfully performed in an attempt to combine resistance to both crown gall and Pratylenchus vulnus nematode into the same rootstock (gene stacking) (Walawage et al 2013). A. tumefaciens was also used to transfer the fld gene from cyanobacteria to J. regia in order to increase osmotic stress tolerance (Sheikh Beig Goharrizi et al 2016).…”

Advances in Understanding the Molecular Mechanisms of Root Lesion Nematode Host Interactions

“…A caveat to using RNAi as a functional tool is that the extent of silencing depends on several factors, including the amount of dsRNA delivered, the sequence chosen, the natural level of target gene expression, whether the target is a member of a multigene family, and whether there is apparent recovery from RNAi phenotypes-these parameters vary with target gene and species (26,102,106). However, the recent demonstration that hairy roots engineered to produce nematode dsRNA can silence the nematode target genes of P. thornei, P. vulnus, and P. penetrans feeding on them is a positive demonstration that host-induced gene silencing could provide an effective alternative to current chemical and conventional control strategies for RLNs (29,106,112,114).…”