DISEASE AND PEST RESISTANT TRANSGENIC ROOTSTOCKS: ANALYSIS, VALIDATION, DEREGULATION AND STACKING OF RNAi-MEDIATED RESISTANCE TRAITS

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1 DISEASE AND PEST RESISTANT TRANSGENIC ROOTSTOCKS: ANALYSIS, VALIDATION, DEREGULATION AND STACKING OF RNAi-MEDIATED RESISTANCE TRAITS Sriema L. Walawage, Russell Reagan, Alice Lee, Michael V. McKenry, Charles A. Leslie, Michael Braverman, Brad Hanson, and Abhaya M. Dandekar ABSTRACT Crown gall disease, caused by the bacterium Agrobacterium tumefaciens, is a significant source of economic losses in walnut orchards and nurseries in California. A crown gall resistant rootstock was developed as a potential solution to this problem using RNAi technology. The crown gall resistant paradox rootstock (CGR-PR) lines were created using the binary vector pde (Escobar et al., 2001; 2002; 2003a and Fig. 1). These transgenic rootstocks were generated by Agrobacterium-mediated plant transformation that express double stranded RNA corresponding to the common and highly conserved Agrobacterium T-DNA encoded genes ipt and iaam responsible for tumor formation (Escobar et al., 2001; 2002; 2003a). The genetically engineered rootstocks are able to block the expression of these genes resulting in the suppression of tumor formation (Escobar et al., 2001; 2002; 2003). Now that the success of this technology has been demonstrated with a transgenic Paradox hybrid genotype, next step is field testing to establish efficacy under rigorous field conditions at multiple locations and eventual commercial release. We initiated and compilation of data to fulfill the requirements for APHIS deregulation and EPA registration. For APHIS deregulation requirements we are compiling information for 1) weediness, 2) gene flow and 3) horticultural characteristics. For Paradox rootstocks in general 1 and 2 should not be a problem for 3) we collecting data from a field trial of CGR-PR rootstock with a scion to evaluate how similar the CGR-PR rootstock is to wild type PR. The transgenic events were planted in field plots in Hutchison and Armstrong fields in Yolo and Solano counties respectively. Here each event grows as either a tree or as a rootstock grafted to a Chandler scion. Elite transgenic events have been identified and include the Paradox lines J1a 20A, J1a 19A, J1a 1A and RR4 1A, 10A, 12A. These were selected as elite rootstocks as they showed the best resistance to tumor formation when infected with a mixture of wild type tumor forming strains of Agrobacterium tumefaciens. As a control wild type Paradox rootstocks (sensitive to Agrobacterium tumefaciens) grafted to Chandler scion variety in the same block. Crown gall resistant lines were analyzed for their horticultural characteristics mainly tree diameter and tree height. We investigated the elite J1a 1A line for its susceptibility to root lesion nematodes and evaluated its allelopathy phenotyle by analyzing the surrounding weed populations and comparing these with non-transgenic control J1a parental line. Experimental data revealed that neither the nematode susceptibility nor the allelopathy phenotype was modified due to the presence of the elite allele in the J1a rootstock. Biochemical analysis of bark samples has been initiated to analyze differences in metabolites. California Walnut Board 73 Walnut Research Reports 2015

2 OBJECTIVES Overall Goal: Develop Paradox rootstocks resistant to disease and pests Objective 1: Analysis and deregulation of crown gall resistant Paradox rootstock (CGR-PR) Activity 1: Molecular analysis of elite lines Metabolome analysis of bark samples from root stock, scion and union junction of trees of J1a 1A and J1a control Activity 2: Initiating the data gathering for deregulation of CGR-PR. a. Evaluating allelopathy phenotype of RNAi-mediated crown gall disease resistance walnut line J1a 1A by investigating surrounding weed populations b. Evaluation of horticultural characteristics for RNAi crown gall walnut line J1a 1A c. Evaluation of resistance to Pratylenchus vulnus for RNAi crown gall walnut line J1a 1A SIGNIFICANT FINDINGS Metabolome analysis of bark samples from root stock, scion and union junction of trees of J1a 1A and J1a control Total of 202 biochemicals were identified from walnut bark The biochemical profiles of J1a 1A varied in various aspects from J1a controls Raffinose and its precursor galactinol, sucrose, glucose and maltose were higher in the rootstock of J1a 1A trees implying a greater amount of carbon was available in these plants Several amino acids such as glutamate and aspartate were lower in the J1a 1A trees compared to J1a controls, reflecting either a higher rate of utilization of these molecules in biosynthesis of cellular components or a limitation of carbon availability due to carbon being directed to raffinose for storage The level of serotonin in the J1a 1A graft union was lower than the J1a control graft union compared to their corresponding root stocks. Accumulation of serotonin in root stocks can be induced in response to necrotic fungal infections. Weediness, horticultural characteristics and susceptibility to root lesion nematodes of crown gall resistant J1a 1A compared to the J1a control Allelopathy phenotype determined by measuring the composition of surrounding grass and broadleaf populations and the susceptibility to walnut root lesion nematode have not changed in the elite line J1a 1A compared to J1a controls. There were no observed differences horticultural characteristics measured by the tree diameters or tree heights of J1a 1A trees compared to J1a control trees. PROCEDURES Objective 1: Analysis and deregulation of crown gall resistant Paradox rootstock (CGR-PR) Activity 1: Metabolome analysis of bark samples from root stock, scion and union junction of trees of J1a 1A and J1a control: Six bark strips that included tissue corresponding to the scion, graft union and rootstock segments were dissected each from three J1a 1A and three J1a control trees growing in the Hutchison field. Once dissected each of the bark strips were further California Walnut Board 74 Walnut Research Reports 2015

3 dissected to separate the scion, graft union and rootstock tissues and then the 18 bark tissue segments were flash frozen immediately in liquid nitrogen. Each bark segment was then ground to a fine powder in liquid nitrogen and then stored frozen at -80C prior to the analysis (Fig.2). To conduct an analysis of the metabolites present in these 18 bark samples ~100 mg of the powdered bark samples were sent to Metabolon Inc to conduct the metabolite analysis. At Metabolon Inc, 20 mg of each bark sample was thawed on ice and extracted using an automated MicroLab STAR system (Hamilton Company) in 400 ul of methanol containing internal standards. UPLC/MS was performed using a Waters Acquity UHPLC (Waters Corporation) coupled to an LTQ mass spectrometer (Thermo Fisher Scientific Inc.) equipped with an electrospray ionization source. Two separate UHPLC/MS injections were performed on each sample: one optimized for positive ions and one for negative ions. Samples used for GC/MS were first chemically derivatized and then analyzed on a Thermo-Finnigan Trace DSQ fastscanning single-quadrupole MS operated at unit mass resolving power. Chromatographic separation, followed by full-scan mass spectra, was performed to record retention time, molecular weight (m/z), and MS/MS2 of all detectable ions present in the samples. Metabolites were identified by automated comparison of the ion features in the experimental samples to a reference library of chemical standard entries that included retention time, molecular weight (m/z), preferred adducts, and in-source fragments, as well as their associated MS/MS2 spectra. This library allowed the rapid identification of metabolites present in the bark tissue samples with high confidence. Comparison of bark samples to process blanks (water only) and solvent blanks allows the removal of artefactual peaks Activity 2: Analysis and deregulation of crown gall resistant Paradox rootstock (CGR-PR). a. Evaluating allelopathy phenotype of RNAi-mediated crown gall disease resistance walnut line J1a 1A by investigating surrounding weed populations: The experiments were conducted at UC Davis field facilities in Solano and Yolo Counties. The purpose of this trial was to determine if walnut trees with RNAi-mediated crown gall disease resistance allele (J1a 1A) displayed any change in their allelopathy phenotype. This was investigated by evaluating the allelopathic impact of transgenic rootstock (J1a-IA) on the composition of surrounding weed populations compared to those surrounding wild type walnut trees (J1a control) that do not express crown gall resistance transgenic allele. We investigated resident weed populations (species and quantity) on the orchard floor in the immediate area of established crown gall resistant and non-resistant walnut trees. Additionally, we will collect soil samples from the root zone of resistant and non-resistant walnut trees in the orchard, extract water soluble compounds with a crude extract, and use the extract in germination assays with several weed and crop species. Transformed and non-transformed trees and their respective surrounding environments were assessed for differential effects on resident weeds populations and on germination of representative annual plants. Two types of analysis were conducted to accomplish this. The density and population composition of resident weeds were evaluated. At evaluation, weeds were counted near the base of the elite, transformed walnut line J1a 1A and the non-transformed J1a control. Weeds were counted and identified to species in a ½ m square area in the tree row where conventional herbicide treatments have been used and in the adjacent alleyway where vegetation is managed with mowing. Population data were analyzed using analysis of variance and appropriate means separation procedures. Weeds in the orchard managed using best management practices for the production region and included use of conventional herbicide programs to minimize weed competition with the test trees. Herbicides California Walnut Board 75 Walnut Research Reports 2015

4 will be applied in strips within the tree row by research station personnel and vegetation between tree rows will be managed with periodic mowing. b. Evaluation of horticultural characteristics for RNAi crown gall walnut line J1a 1A The crown gall resistant Paradox rootstock (CGR-PR) lines currently planted in field plots located in Solano and Yolo counties include the following elite transgenic events, Paradox lines J1a 20A, J1a 19A J1a 1A and RR4 1A, 10A, 12A. These trees are present in these locations as un-grafted trees and as grafted trees where the scion is the Chandler variety. Crown gall resistant lines were analyzed for their horticultural characteristics measuring tree diameter and tree height. Tree circumference data were collected in 2014 and 2015 from Armstrong and Hutchison fields located in Solano and Yolo counties respectively. Tree girth measurements were made at 24" from base of tree (soil level). Tree height data were collected in 2015 from Armstrong field. Tree diameter and height means of each transgenic line were compared with non-transgenic controls using Student s t-test. P-values less than 0.01 were classified as significantly different from controls and denoted by a whereas and those greater than 0.01 were as not significantly different to the controls and denoted by b. c. Evaluation of resistance to walnut root lesion Pratylenchus vulnus for RNAi crown gall walnut line J1 1A In vitro rooted walnut micro-shoots of the transformed cultivar J1a 1A and non-transformed J1 a control were tested for evaluation of resistance/sensitivity to the walnut rootlesion nematode (RLN; Pratylenchus vulnus). Micro-shoots were transferred to culture tubes containing 30 ml DKW medium to initiate root formation. A rapid nematode resistance screening assay was used to test nematode multiplication in transformed roots. Initially roots were inoculated with 100 nematodes isolated under in vitro conditions. Nematodes were allowed to feed on roots for two months. After two months of co-cultivation, the total nematode population in each tube was isolated using Baermann funnels. There were four biological replicates from each of the transgenic line and control line. The entire experiment was repeated twice at different times. The numbers of nematodes present in each tube was calculated and final nematode counts were obtained. In vitro procedures used for this analysis were explained in detail in Walawage et al., (2013). Nematode population means of transgenic line J1a 1A was compared with nontransgenic control J1a control using Student s t-test. RESULTS AND DISCUSSION Metabolome analysis of bark samples from root stock, scion and union junction of trees of J1a 1A and J1a control: Two hundred and two biochemicals were detected in the 18 bark tissue samples. Pair wise comparisons were made of the metabolite profiles present in each tissue sample by comparing the similar tissues between the transgenic vs control bark strips. The biochemical profiles of trees grafted with root stock from transgenic trees (J1a 1A) varied in various aspects from that of trees grafted with root stock from WT Trees (J1a controls). In particular, in transgenic trees raffinose and its precursor galactinol were elevated. Sucrose, glucose and maltose were higher in the rootstock of transgenic plants implying a greater amount of carbon was available in these plants. Several amino acids, particularly glutamate and aspartate which are the entry point of carbon and nitrogen into many biomolecules such as co-factors and nucleic acids, were lower in the transgenic trees compared to WT, reflecting either a higher rate of utilization of these molecules California Walnut Board 76 Walnut Research Reports 2015

5 in biosynthesis of cellular components or a limitation of carbon availability due to carbon being directed to raffinose for storage or as a compatible solute for stress mitigation. The level of serotonin in the transgenic graft union was fold lower than the WT graft union reflecting the serotonin level of the corresponding rootstocks. Serotonin was below limit of detection in the scion tissue. The data for each biochemical displayed as box plots like that shown in the example Fig.3. These differences are un-related to the transgene and likely represent natural variation that occurs during the tissue culture and regeneration process. Evaluating allelopathy phenotype of RNAi-mediated crown gall disease resistance walnut line J1a 1A by investigating surrounding weed populations We investigated resident weed populations (species and quantity) on the orchard floor in the immediate area of established crown gall resistant and comparable non-resistant walnut trees. Transformed and non-transformed trees and their respective surrounding environments were assessed for differential effects on resident weeds populations and on germination of representative annual plants. The density and population composition of resident weeds were evaluated. At evaluation, weeds were counted near the base of the elite, transformed walnut line J1a 1A and its nontransformed J1a control. Weeds were counted and identified to species in a ½ m square area in the tree row where conventional herbicide treatments have been used and in the adjacent alleyway where vegetation is managed with mowing. Population data were analyzed using analysis of variance and appropriate means separation procedures. Weeds in the orchard managed using best management practices for the production region and included use of conventional herbicide programs to minimize weed competition with the test trees. Herbicides apply in strips within the tree row by research station personnel and vegetation between tree rows manage with periodic mowing. According to the T-tests, no statistical differences were observed in surrounding weed populations. The initial weed counts from July 2015 are shown in Fig. 4 and the total grass and broadleaf weeds per square meter (+ SE) in the unsprayed area adjacent to transgenic (J1a 1A) or the non-transformed control trees (J1a control) are shown in Fig. 5. Evaluation of horticultural characteristics for RNAi crown gall walnut line J1a 1A Fig. 6 shows the map of Hutchison field in Yolo County, CA and Fig. 7 shows the same field in winter season Hutchison field was established in 2008 with 154 total trees. There are 29 controls, 125 transgenic rootstock lines, 73 budded with Chandler, 7 J1a-1A trees, 3 J1a-1A budded to Chandler and 3 J1a controls. The boxes highlighted in yellow indicate the budded trees to Chandler and boxes highlighted in grey indicate buffer trees. All the J1a-1A budded trees are marked in green line color and all the budded J1a controls are marked in blue line color. Armstrong field was established in 2013 with 96 plants. Currently there are 86 plants as 10 trees died. This particular field has ten J1a 1A and eleven J1a controls. Table 2 shows the tree diameter data for J1 and RR4 lines in 2014 and 2015 in the Armstrong and Hutchison fields. These data revealed that there are no significant differences observed in tree diameter compared to the controls in any transgenic lines in any of the fields in any of the years. Fig. 8 and Fig. 9 show the tree height analysis in J1 and RR4 transgenic lines compare to non-transgenic J1a controls and RR4 controls in 2015 in Armstrong field (after two years of field establishment). P-values less than 0.01 are significantly different from controls and denoted by a whereas and greater than 0.01 are not significant different to controls and denoted by b. These data revealed that there are some differences in tree height in J1a 1A and J1a 20A compared to the controls. This difference was not observed in RR4 lines after two years of planting. California Walnut Board 77 Walnut Research Reports 2015

6 Evaluation of resistance to Pratylenchus vulnus for RNAi crown gall walnut line J1a 1A In vitro rooted walnut micro-shoots of the transformed cultivar J1a 1A and non-transformed J1 a control were tested for evaluation of resistance to Pratylenchus vulnus (Fig. 10A). A rapid screening method was used to test for nematode resistance in RNAi crown gall walnut line J1a 1A and the J1a control. Cultures initiated using 100 nematodes per rooted plant. After two months of co-cultivation, the total nematode population in each tube was isolated using Baermann funnels (Fig. 10B). Results of these trials are shown in the Fig.11 and Fig. 12. Nematode infestation is expressed as the number of nematodes recovered from cultures Nematodes were recovered per root for transgenic line J1a 1A and J1a controls after two months of in vitro co-culture in the dark. Bars represent mean of four replicates (Error bars=s.d). There were no significant differences observed in nematode populations in transgenic line J1a 1A compared to un-transformed J1a controls. Comparisons were considered significant whenever p<0.05. REFERENCES Escobar, M.A., E.L. Civerolo, K.R. Summerfelt, and A.M. Dandekar RNAi-mediated oncogene silencing confers resistance to crown gall tumorigenesis. In: Proceedings of the National Academy of Sciences of the United States of America. 98(23): Escobar, M.A., C.A. Leslie, G.H. McGranahan and A.M. Dandekar Silencing crown gall disease in walnut (Juglans regia L.). Plant Sci. 163(3): Escobar, M.A., E.L. Civerolo, V.S. Polito, K.A. Pinney and A.M. Dandekar. 2003a. Characterization of oncogene-silenced transgenic plants: Implications for Agrobacterium biology and posttranscriptional gene silencing. Molecular Plant Pathology 4(1): Escobar, M.A., and A.M. Dandekar. 2003b. Agrobacterium tumefaciens as an agent of disease. Trends Plant Sci Aug;8(8): Walawage SL, Britton MT, Leslie CA., Uratsu, SL, Li YY, Dandekar AM: Stacking resistance to crown gall and nematodes in walnut rootstocks. BMC Genomics, 2013, 14: 668 California Walnut Board 78 Walnut Research Reports 2015

7 Table 1: 18 tissue samples collected from grafted walnut trees. Bark Samples GROUP_DESCRIPTION Position J1 1A-S1 iaam/ipt Scion Scion J1 1A-S2 iaam/ipt Scion Scion J1 1A-S3 iaam/ipt Scion Scion J1 1A-U1 iaam/ipt Graft Union Graft Union J1 1A-U2 iaam/ipt Graft Union Graft Union J1 1A-U3 iaam/ipt Graft Union Graft Union J1 1A-R1 iaam/ipt Rootstock Rootstock J1 1A-R2 iaam/ipt Rootstock Rootstock J1 1A-R3 iaam/ipt Rootstock Rootstock J1 C-S1 WT Scion Scion J1 C-S2 WT Scion Scion J1 C-S3 WT Scion Scion 1 C-U1 WT Graft Union Graft Union J1 C-U2 WT Graft Union Graft Union J1 C-U3 WT Graft Union Graft Union J1 C-R1 WT Rootstock Rootstock J1 C-R2 WT Rootstock Rootstock J1 C-R3 WT Rootstock Rootstock Fig. 2: Chandler scion grafted to transgenic paradox rootstock just before bark tissues were removed California Walnut Board 79 Walnut Research Reports 2015

8 Box Plot Legend Extreme Data Point Metabolite Name Max of distribution 1.3 Scaled Intensity J1C-R J1C-S J1C-U J1 1A-R J1 1A-S J1 1A-U Walnut Bark Group Wild Type, Rootstock Wild Type, Scion Wild Type, Graft Union Transgenic, Rootstock Transgenic, Scion Transgenic, Graft Union Limit of Upper Quartile Median Value Mean Value Limit of Lower Quartile Min of distribution Fig. 3: Analysis of metabolites in bark tissues. J1C-R-WT rootsctock, J1C-S-WT scion, J1C-U-WT graft union, J11A-R-Transgenic rootstock, J11A-S-Transgenic scion, J11A-U-Transgenic graft union Fig. 4: Average grass and broad leaf per square meter in the unsprayed area adjacent to J1a controls and J1a 1A trees Fig 5: Weed density of grasses and broadleaf surrounding J1a controls and J1 1A trees California Walnut Board 80 Walnut Research Reports 2015

9 Fig. 6: Field map of Hutchison field, Yolo County, CA. This field was established in 2008 with 154 total trees. There are 29 controls, 125 transgenic rootstock lines, 73 budded with Chandler, 7 J1a-1A trees, 3 J1a-1A budded to Chandler and 3 J1a controls. The boxes highlighted in yellow indicate the budded trees to Chandler and boxes highlighted in grey indicate buffer trees. All the J1a-1A budded trees are marked in green line color and all the budded J1a controls are marked in blue line color. Fig 7: Hutchison field, Yolo County in winter Trees planted for transgenic walnut rootstock trail. California Walnut Board 81 Walnut Research Reports 2015

10 Table 2: Tree diameter analysis of crown gall transgenic and non-transgenic genotypes in Armstrong and Hutchison fields Genotype J1 A control Number of trees Armstrong * Hutchison ** Mean Mean Number Number diameter diameter of trees of trees (cm) (cm) Mean diameter (cm) J1 1A b b b J1 19A b b b J1 20A a b N.A. N.A. RR4 control RR4 1A b b b RR4 10A b b b RR4 12A b b b * Trees were planted on 2013, ** Trees were planted on Circumferences were taken at 24" from base of tree. P-values less than 0.01 are significantly different from controls and denoted by a whereas and greater than 0.01 are not significant different to controls and denoted by b. N.A.-data not available Tree height (m) a b a J1 a control J1a 1A J1a 19A J1a 20A Transgenic line Fig. 8: Tree height analysis in J1 transgenic lines compare to non-transgenic J1a control in 2015 in Armstrong field (after two years of field establishment). P-values less than 0.01 are significantly California Walnut Board 82 Walnut Research Reports 2015

11 different from controls and denoted by a whereas and greater than 0.01 are not significant different to controls and denoted by b. (Error bars=s.d) Fig. 9: Tree height analysis in RR4 transgenic lines compare to non-transgenic RR4 control in 2015 in Armstrong field (after two years of field establishment). P-values less than 0.01 are significantly different from controls and denoted by a whereas and greater than 0.01 are not significant different to controls and denoted by b. (Error bars=s.d) Fig. 10: (A) Rooted J1a control and J1a 1A plants ready to innoculate nematodes. (B) Nematode isolation using Baermann funnels. California Walnut Board 83 Walnut Research Reports 2015

12 Fig. 11: Number of root lesion nematodes present in each replicate of J1a controls and J1a 1A transgenic lines after two months of inoculation. Fig. 12: Number of root lesion nematodes presented in roots of J1a control and J1a 1A roots after two months of innoculation. Nematode infestation is expressed as the number of nematodes recovered from cultures initiated using 100 nematodes per rooted plant. Nematodes were recovered per root for transgenic line J1a 1A and J1a control after two months of in vitro co-culture in the dark. Bars represent mean of four replicates (Error bars=s.d). No significant differences from controls are denoted with b. Comparisons are considered significant whenever p<0.05. California Walnut Board 84 Walnut Research Reports 2015