Enhanced resistance to Rhizoctonia solani by combined expression of chitinase and Ribosome Inactivating Protein in transgenic potatoes (Solanum tuberosum L.)

Abstract

Potato (Solanum tuberosum L.) is susceptible to many fungal pathogens including Rhizoctonia solani. In the present study, the potato cultivar Desirée was transformed via Agrobacterium tumefaciens strain GV3101 containing the binary plasmid pGJ132 harboring both the chitinase (chiA) and rip30 genes. The potato leaf disc was used as an explant for transformation. PCR, Southern blot and Western blot were used for characterization of the transgenic plants. In this study it was shown that not all the plants developed in selective medium were positive for the corresponding gene using the PCR technique. Southern blot analysis confirmed that transgenic plants integrated 2-3 copies of chiA and rip30 genes respectively into their genome. The expression of the CHIA and RIP30 proteins was confirmed in the leaf extracts of the transgenic clones by Western blot analysis. Transgenic potato plants expressing rip30 and chiA genes showed enhanced resistance to R. solani in a greenhouse assay.Additional key words: fungal resistance; genes; proteins; southern blot; western blot. ResumenResistencia aumentada a Rhizoctonia solani por la expresión combinada de quitinasa y proteínas inactivantes de los ribosomas en patatas transgénicas La patata (Solanum tuberosum L.) es susceptible a muchos hongos fitopatógenos, incluyendo Rhizoctonia solani. En el presente estudio, se transformó el cultivar de patata 'Desirée' mediante Agrobacterium tumefaciens, cepa GV3101, que contiene el plásmido binario pGJ132 que alberga los genes quitinasa (chiA) y rip30. Se utilizaron discos de hojas como explante para la transformación de plantas. Se utilizaron las técnicas de PCR, Southern y Western blot para la caracterización de las plantas transgénicas. En este estudio se demostró, mediante PCR, que no todas las plantas que se desarrollaron en medio selectivo fueron positivas para el gen correspondiente. El análisis de Southern blot confirmó que las plantas transgénicas integraron en su genoma 2-3 copias de los genes chiA y rip30. Se llevó a cabo un ensayo de invernadero para evaluar la resistencia a R. solani de los clones transgénicos que expresan los transgenes. Las plantas transgénicas que expresan los genes rip30 y chiA mostraron una resistencia completa a R. solani.

Antifungal Activity of Ribosome-Inactivating Proteins

“…To enhance the antifungal activity of RIPs, genes encoding chitinases have also been introduced [79,84,86,99,110,111], which as we have seen exert a synergistic effect with…”

“…To enhance the antifungal activity of RIPs, genes encoding chitinases have also been introduced [ 79 , 84 , 86 , 99 , 110 , 111 ], which as we have seen exert a synergistic effect with RIPs, and even the lectin WGA [ 99 ] ( Figure 2 ). In addition, some transgenic plants carrying RIPs are also resistant to viruses and insects [ 9 , 12 ], which adds even more interest to this type of strategy.…”

Extensive Evolution of Cereal Ribosome-Inactivating Proteins Translates into Unique Structural Features, Activation Mechanisms, and Physiological Roles

“…Transformed Indian mustard ( Brassica juncea ) expressing both barley RIP and chitinase showed 44% reduction in Alternaria brassicae hyphal growth in an in vitro assay and transgenic plants sprayed with an A. brassicae spore suspension showed reduced numbers, sizes, and expansion of lesions compared to wild type plants [ 133 ]. Similarly, potato plants overexpressing a combination of RIP and chitinase showed enhanced resistance to Rhizoctonia solani in a greenhouse assay [ 134 ]. Chopra and Saini [ 135 ] reported that transformed blackgram ( Vigna mungo ) plants expressing the same set of genes arrested the growth of Corynespora cassiicola in an in vitro antifungal assay and the plants also showed less disease symptoms compared to wild type plants after being sprayed with a spore suspension.…”