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F.A. Bliss, P.L. Schuerman, A.A. Almehdi, A.M. Dandekar and N. Bellaloui

Crown gall is an important disease of many fruit and nut crops, but little is known about sources of resistance. We screened germplasm from Prunus armeniaca L., P. angustifolia Marsh., P. argentia L., P. avium L., P. besseyi Bailey, P. bokhariensis Schneid., P. brigantica L., P. cerasifera Ehrh., P. cerasus L., P. dulcis (Mill.) D.A. Webb, P. fruiticosa Pall., P. hortulana Bailey, P. insititia L., P. japonica Thunb., P. mahaleb L., P. persica (L.) Batsch, P. serotina Ehrh., P. simonii Carr., P. sogdiana L., and P. webbii (Spach) Vieh. When either main stems or lateral branches of seedlings were inoculated with strains K12 and C58 of Agrobacterium tumefaciens (Smith and Townsend) Conn., the incidence of resistance was less than 10% except in some accessions of P. mahaleb L. where up to 30% of the plants were resistant. Some resistant plants were identified in other species, with P. insititia L. being the most promising. Symptoms based on presence and size of galls should be allowed to develop for up to 90 days after inoculation to reduce the likelihood of misclassifying plants as resistant when they are slightly susceptible.

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Matthew A. Escobar, Andrew Shilling, Pine Higgins, Sandra L. Uratsu and Abhaya M. Dandekar

The enzyme polyphenol oxidase (PPO) is nearly ubiquitous in Kingdom Plantae and catalyzes the oxidation of phenolic compounds into highly reactive quinones. Although the functional importance of PPO in plants remains uncertain, a putative antipathogen role for walnut (Juglans regia) PPO was posited as early as 1911. However, despite the rich diversity of phenolics present in walnut leaves and hulls, walnut PPO has been little studied since the early 1900s. We cloned a PPO-encoding gene from a walnut pistillate flower cDNA library and designated the gene jrPPO1. Genomic Southern analysis demonstrated that jrPPO1 is the sole PPO gene in walnut. Transgenic tobacco (Nicotiana tabacum) plants expressing jrPPO1 display greater than 10-fold increases in leaf PPO activity compared with wild-type tobacco, demonstrating that jrPPO1 encodes a functional enzyme. The jrPPO1 protein is expressed primarily in the leaves, hulls, and flowers of walnut trees and is not regulated by wounding or methyl jasmonate. To examine whether walnut PPO could affect pathogen resistance, tobacco plants expressing jrPPO1 were challenged with Pseudomonas syringae pv. tabaci. Based on both symptom development and quantitative analyses of bacterial growth in planta, the PPO-expressing plants did not display increased resistance to this pathogen. Leaf extract browning assays indicated that tobacco leaves lack the endogenous phenolic substrates required for significant jrPPO1 activity and quinone production in planta.

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R. Tao, H. Yamane, H. Sassa, H. Mori, H. Murayama, T.M. Gradziel, A.M. Dandekar and A. Sugiura

Stylar proteins of four Prunus species, P. avium, P. dulcis, P. mume, and P. salicina, were surveyed by 2D-PAGE combined with immunoblot and N-terminal amino acid sequence analyses to identify S-proteins associated with gametophytic SI in the Prunus. All four S-allelic products tested for P. dulcis could be identified in the highly basic zone of the gel. These S-proteins had Mr of about 28–30 kDa and reacted with the anti-S4-serum prepared from Japanese pear (Pyrus serotina). Two of six S-allelic products tested for P. avium could be also identified in the 2D-PAGE profiles, with roughly the same pI and Mr as those of S-proteins of P. dulcis. Putative S-proteins for P. mume and P. salicina were found in the same area of 2D-PAGE as the area where S-proteins of P. avium and P. dulcis were located. N-terminal amino acid sequence analysis of these proteins revealed that they were similar to S-RNases reported previously.

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G.C. Martin, A.N. Miller, L.A. Castle, J.W. Morris, R.O. Morris and A.M. Dandekar

The udiA gene encoding the enzyme β -glucuronidase (GUS) appears promising as a genetic marker for early confirmation of successful plant cell transformation. Two strains of Agrobacterium rhizogenes and eight strains of A. tumefaciens were selected as hosts to carry a binary plasmid (pBI121) containing the marker gene encoding the GUS marker that is controlled by the CaMV35S promoter. Presence of plasmid pBI121 in the bacteria was confirmed by resistance to kanamycin, plasmid re-isolation, and restriction enzyme analysis. When the GUS enzyme was expressed in transformed plant cells, reaction with the histochemical substrate 5-bromo-4-chloro-3-indolylglucuronide (X-gluc) lead to blue pigment development. Expression of GUS in viable bacteria that had not been eliminated from transformed tissue caused problems with the early transformation detection in radish, peach, and apple stem sections by also producing a positive X-gluc color reaction. Putative transformation of apple xylem parenchyma callus was accomplished, as judged by resistance to kanamycin, opine analysis, GUS marker gene expression, and presence of the APH(3')11 enzyme. In this system, elimination of bacterial contamination was accomplished during multiple culture transfers on selective media. To be more useful as a marker, the GUS gene should be coupled with a promoter that will not be expressed by Agrobacterium. Parenchyma callus may serve as a primary screen to provide an efficient way of determining the ideal strain for transformation of deciduous tree fruit genera. In our studies, strain A281 consistently proved to be a vector superior to others tested.

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Fatemeh Khodadadi, Masoud Tohidfar, Mehdi Mohayeji, Abhaya M. Dandekar, Charles A. Leslie, Daniel A. Kluepfel, Timothy Butterfield and Kourosh Vahdati

Plants respond to pathogens with both active and passive defense mechanisms. These defense responses include the induction of defense or defense-related genes such as polyphenol oxidase (PPO) and pathogenesis-related (PR) proteins. The role of PPO in the interaction between bacterial blight [Xanthomonas arboricola pv. juglandis (Xaj)] and walnut (Juglans regia) was studied. JrPPO-1 and P14a genes were identified in two walnut cultivars, Chandler and Serr, using standard polymerase chain reaction (PCR) to understand their inducible ability in response to Xaj. ‘Serr’ and ‘Chandler’ were inoculated with Xaj strain 417. PPO activity in leaves was assayed at 0, 24, 72, 96, 120, and 144 hours after inoculation. Results showed a steady increase in activity commencing within 24 hours of inoculation. Increase in PPO activity was close to 2-fold greater in ‘Chandler’ than in ‘Serr’ at all time points examined. Real-time PCR analysis showed differences between cultivars in PPO gene expression. The JrPPO-1 gene was highly expressed in both cultivars 24 hours after inoculation but expression in ‘Serr’ was much greater than in ‘Chandler’. Significant expression of P14a gene was observed in both cultivars within 24 hours. Expression in ‘Serr’ was strong and maximized with a significant increase at 96 hours. Expression in ‘Chandler’ was far weaker than ‘Serr’ at 24 hours and did not increase further. Our results imply that the walnut–bacterial blight interaction induces the expression of JrPPO-1 and P14a as well as the activity of PPO.

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Kourosh Vahdati, James R. McKenna, Abhaya M. Dandekar, Charles A. Leslie, Sandie L. Uratsu, Wesley P. Hackett, Paola Negri and Gale H. McGranahan

Walnuts (Juglans spp.) are difficult-to-root woody plants. The rolABC genes (rolA + rolB + rolC), derived from the bacteria Agrobacterium rhizogenes, have been shown to increase the rooting potential of other difficult-to-root woody plants. We inserted the rolABC genes into somatic embryos of a `Paradox' hybrid (J. hindsii × J. regia) clone PX1 using the A. tumefaciens gene transfer system. A transgenic sub-clone, designated PX1 rolABC 2-2 was selected and compared to the untransformed clone for a variety of phenotypic characteristics, including rooting potential. Transformed and untransformed shoots were budded onto seedling J. regia rootstock in the greenhouse and established in the field. Transformed trees displayed reduced internode length, an increase in lateral branching, and wrinkled leaves. In another test, a commercial persian walnut cultivar J. regia `Chandler' was grafted onto rooted cuttings of both the untransformed and transformed plants. The presence of the rolABC genes in the rootstock had no visible effects on the grafted scion. Several of these trees were excavated from the field and the root systems of each genotype were examined for root number, diameter, and biomass. Trees with the rolABC rootstock had significantly more small diameter roots compared to the controls and less recovered biomass. Tests of the rooting potential of leafy semi-hardwood cuttings for two years resulted in 14% to 59% rooting of the transformed cuttings compared to 51% to 81% rooting of the control. Both transformed hardwood cuttings and microshoots in tissue culture also rooted significantly less (52% and 29% respectively) than untransformed hardwood cuttings and tissue cultured shoots (82% and 54% respectively). Thus, although the rolABC genes induced a shorter internode length and a more fibrous root system (typical of rol-tranformed plants), they were not useful for increasing the rooting potential, and as rootstock they did not affect the phenotype of the scion.

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A.M. Dandekar, H.J. Fisk, G.H. McGranahan, S.L. Uratsu, H. Bains, C.A. Leslie, M. Tamura, M. Escobar, J. Labavitch, C. Grieve, T. Gradziel, P.V. Vail, S.J. Tebbets, H. Sassa, R. Tao, W. Viss, J. Driver, D. James, A. Passey and G. Teo