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.

Cotton being the major fiber crop across the world is exposed to numerous biotic and abiotic stresses. Genetic transformation of cotton is vital to meet the world's food, feed and fiber demands. Genetic manipulation by randomly transferring the genes emanate variable gene expression. Targeted gene insertion by latest genome editing tools results in predictable expression of genes at a specified location. Gene stacking technology emerged as an adaptive strategy to combat biotic and abiotic stresses by integrating 2-3 genes simultaneously and at a specific site to avoid variable gene expression at diverse locations. This study explains the development of cotton's founder transformants to be used as a base line for multiple gene stacking projects. We introduced Cre and PhiC31 mediated recombination sites to specify the locus of incoming genes. CRISPR-Cas9 gene was integrated for developing CRISPR based founder lines of cotton. Cas9 gene along with gRNA was integrated to target Rep (replication) region of cotton leaf curl virus. Replication region of virus was specifically targeted to diminish further proliferation and preventing the virus to develop new strains. To successfully develop these primary transformants, a model transformation system has been optimized with the red color visualization (DS-Red). Following red color transformation system, three baselines with recombination specified site (Rec), targeted replication region (Rep) and Cas9 founder lines have been developed. These founder transformants are useful for developing recombinase mediated and CRISPR/Cas9 based originator lines of cotton. Moreover, these transformants will set up a base system for all the recombinase mediated gene stacking projects.

Crown gall disease (Agrobacterium tumefaciens), crown/root rot disease (Phytophthora spp.), root lesion disease (Pratylenchus vulnus) and tree vigor are key traits affecting the productivity and quality of walnuts in California. Unchallenged hybrid rootstocks were analyzed by RNA-seq to examine pre-formed factors affecting these traits. Enrichment analysis of the differentially expressed genes revealed that the increased expression of cell wall biogenesis-related genes plays a key role in susceptibility to A. tumefaciens, susceptibility to Phytophthora spp. and increased vigor. Analysis of the predicted subcellular loci of the encoded proteins revealed that many gene products associated with vigor and susceptibility were targeted to the plasma membrane and extracellular space, connecting these traits to sustaining barrier function. We observed that RNA processing and splicing, along with predicted nuclear targeting, were associated with resistance to A. tumefaciens, resistance to Phytophthora spp. and low vigor. Four genes within the J. microcarpa QTL region for resistance to A. tumefaciens and Phytophthora spp. were represented among our transcripts, with two of the genes being differentially expressed in association with resistance to A. tumefaciens and decreased vigor. No differential expression related to Phytophthora spp. or P. vulnus resistance was observed in this region. Additionally, the J. microcarpa haplotype expressed more transcripts associated with resistance to A. tumefaciens, Phytophthora spp. and low vigor, but not P. vulnus, than the J. regia haplotype. We also report unique and shared hormone and defense responses associated with each trait. This research suggests a link between cell wall biogenesis, vigor and critical root diseases of walnut.

Following photosynthesis, sucrose is translocated to sink organs, where it provides the primary source of carbon and energy to sustain plant growth and development. Sugar transporters from the SWEET (sugar will eventually be exported transporter) family are rate-limiting factors that mediate sucrose transport across concentration gradients, sustain yields, and participate in reproductive development, plant senescence, stress responses, as well as support plant-pathogen interaction, the focus of this study. We identified 25 SWEET genes in the walnut genome and distinguished each by its individual gene structure and pattern of expression in different walnut tissues. Their chromosomal locations, cis-acting motifs within their 5' regulatory elements, and phylogenetic relationship patterns provided the first comprehensive analysis of the SWEET gene family of sugar transporters in walnut. This family is divided into four clades, the analysis of which suggests duplication and expansion of the SWEET gene family in Juglans regia. In addition, tissue-specific gene expression signatures suggest diverse possible functions for JrSWEET genes. Although these are commonly used by pathogens to harness sugar products from their plant hosts, little was known about their role during Xanthomonasarboricola pv. juglandis (Xaj) infection. We monitored the expression profiles of the JrSWEET genes in different tissues of "Chandler" walnuts when challenged with pathogen Xaj417 and concluded that SWEET-mediated sugar translocation from the host is not a trigger for walnut blight disease development. This may be directly related to the absence of type III secretion system-dependent transcription activator-like effectors (TALEs) in Xaj417, which suggests different strategies are employed by this pathogen to promote susceptibility to this major aboveground disease of walnuts.