Robust Response to Plum pox virus Infection via Plant Biotechnology

Abstract

Our goal was to target silencing of the Plum pox virus coat protein (PPV CP) gene independently expressed in plants. Clone C-2 is a transgenic plum expressing CP. We introduced and verified, in planta, the effects of the inverse repeat of CP sequence split by a hairpin (IRSH) that was characterized in the HoneySweet plum. The IRSH construct was driven by two CaMV35S promoter sequences flanking the CP sequence and had been introduced into C1738 plum. To determine if this structure was enough to induce silencing, cross-hybridization was made with the C1738 clone and the CP expressing but PPV-susceptible C2 clone. In total, 4 out of 63 clones were silenced. While introduction of the IRSH is reduced due to the heterozygous character in C1738 plum, the silencing induced by the IRSH PPV CP is robust. Extensive studies, in greenhouse containment, demonstrated that the genetic resource of C1738 clone can silence the CP production. In addition, these were verified through the virus transgene pyramiding in the BO70146 BlueByrd cv. plum that successfully produced resistant BlueByrd BO70146 × C1738 (HybC1738) hybrid plums.

The Use of Transgenic Resistant Plums in Transgrafting Fails to Confer Plum Pox Virus Resistance in Prunus

“…Two methods for transmitting PPV to woody perennials are known: natural aphid transmission and artificial inoculation through grafting. The latter is routinely used in research on the PPV inoculation of woody perennials [22,23,42]. Viable pathogens like PPV that can survive in bud sticks can spread through the vascular system of whole plants; likewise, any molecules in the rootstock, like virus particles or viral RNA, can reach the scion similarly (Tables 2-6, Figure 1).…”

“…Related studies with transgenic cherry trees were successful had siRNAs that moved through the grafting point to confer virus resistance to the targeted host [14]. Having restricted our observations to uninfected healthy plants to look at which tissues were silenced, we did not study the post-transcriptional gene silencing machinery [17,30,33,42] (Figure 4, Figure 5). The root, vascular, periderm and shoot apical meristem tissues seemed differentially methylated (Figure 5B), likely related to their developmental stages [18,22,23,42].…”

Stability of Transgene Inheritance in Progeny of Field-Grown Pear Trees over a 7-Year Period

“…The inheritance of a transgene in tree progeny was first shown in Greensleeves apple trees with the nos and nptII marker genes, where the progeny of two lines showed segregation ratios of 1:1 and 3:1 [23]. Mendelian inheritance of other transgenes at a 1:1 ratio was later shown in apple [31] and plum [32,33]. Mendelian segregation in progeny was also noted in forest trees: poplar with the genes Cry1Ac and nptII demonstrated the ratios of 1:1 and 3:1 [16].…”

“…A study of segregation in T1-T3 generations of tobacco plants with the nptII gene also used 50-100 seeds per line [45]. Previously, researchers studied uidA gene inheritance in the progeny of fruit trees through histochemical staining of seedling leaves [2,32,33] but not seeds. We have demonstrated this method's suitability for evaluating the segregation patterns in progeny.…”

“…It is well known that unintended effects in transgenic plants can be caused by changing environmental conditions or biotic and abiotic stressors [30], and seed quality can indicate these changes. Inheritance of transgenes in progeny has been investigated in earlier studies in apple [26,31] and plum trees [32,33]. Pear is another valuable fruit and is used in genetic engineering to transfer various valuable traits.…”