The Gastrodia antifungal protein (GAFP) is a monocot mannose-binding lectin isolated from the Asiatic orchid Gastrodia elata. This lectin has previously been shown to provide increased resistance in transgenic Nicotiana tabacum against taxonomically unrelated root pathogens Phytophthora nicotianae, Rhizoctonia solani, and Meloidogyne incognita, but its potential to confer disease resistance in tree species is not known. Agrobacterium tumefaciens-mediated transformation yielded three gafp-1 expressing plum lines (Prunus domestica) designated 4J, 4I, and 5D. These lines possessed one, two, and four copies of the gafp-1 gene, respectively, as demonstrated by DNA blotting. Lines 4J and 4I were not phenotypically different from the nontransformed control line, but line 5D showed significant divergence in leaf morphology and growth habit. Compared with the inoculated control line, lines 4J and 4I exhibited increased tolerance to Phytophthora root rot (PRR) caused by P. cinnamomi. When inoculated with the root-knot nematode, Meloidogyne incognita, the 4J and 4I lines showed a significantly lower degree of root galling than the inoculated control line. Nematode reproduction, as measured by the presence of egg masses and the number of eggs produced per gram fresh root, was significantly reduced in line 4J compared with the inoculated control line. The results of this study suggest that the expression of gafp-1 in the roots of a woody plant may confer some level of resistance to PRR and root-knot nematode. Long-term field trials will be necessary to confirm this hypothesis.
Alexis K. Nagel, Guido Schnabel, Cesar Petri and Ralph Scorza
Hetal M. Kalariya, Guido Schnabel, Cesar Petri and Ralph Scorza
The Gastrodia antifungal protein (GAFP-1) is a mannose-binding lectin that can confer increased disease resistance in transgenic tobacco and plum. In all previously generated, transgenic lines, the gene was under the control of the 35SCaMV promoter. In this study, transgenic plum lines were created from seeds derived from open pollination of the cultivar Bluebyrd (BB-OP) with gafp-1 under the control of the polyubiquitin promoter bul409 and evaluated for Phytophthora root rot (PRR) and Root knot nematode (RKN) susceptibility. One of nine transgenic lines synthesizing GAFP-1 exhibited increased tolerance to PRR caused by P. cinnamomi. The same line (BB-OP-1) was also significantly more tolerant to RKN infection caused by Meloidogyne incognita. BB-OP-1 was more resistant to PRR and equally resistant to RKN compared with the cultivar Stanley-derived 4J line, which expresses gafp-1 under the control of the 35SCaMV promoter. GAFP-1 synthesis in BB-OP-1 was not elevated by pathogen infection, suggesting that the bul409 promoter is not inducible in the plum/GAFP-1 system. This study confirms the usefulness of the gafp-1 gene in various cultivars of transgenic plum and establishes that the bul409 promoter is at least equal in effectiveness to the 35SCaMV promoter for gafp-1 expression in transgenic lines of woody plants.
Jean-Michel Hily, Michel Ravelonandro, Vern Damsteegt, Carole Bassett, Cesar Petri, Zongrang Liu and Ralph Scorza
Constructs with self-complementary sequences separated by an intron produce “hairpin” RNA [intron-hairpin-RNA (ihpRNA)] structures that efficiently elicit posttranscriptional gene silencing (PTGS). In the current study, the authors use this technology to confer resistance to plum pox virus (PPV) in herbaceous and woody perennial plants by silencing the PPV–coat protein (CP) gene. The authors confirmed the high capacity of ihpRNA constructs for inducing RNA silencing in Nicotiana benthamiana Domin., as more than 75% of the transformants displayed PTGS as evaluated by specific small interfering RNA (siRNA) production. The authors demonstrated that ihpRNA constructs provided PPV resistance, and they found a correlation between the length of the PPV sequence introduced in the ihpRNA constructs and the frequency of transgenic-resistant plants. Plants transformed with the full-length sequence produced a higher percentage of resistant lines. The authors further demonstrated for the first time that ihpRNA technology is applicable to a woody perennial species. A transgenic plum (Prunus domestica L.) PPV-CP ihpRNA line showed gene silencing characteristics (hypermethylation of the transgene sequence and specific siRNA production) and resistance to PPV infection 16 months after inoculation.