Silencing of cellulose synthase gene(s) of Phytophthora infestans for conferring late blight resistance in Indian potato (Solanum tuberosum L.) cultivar(s)

Abstract

Late blight is the most devastating disease of potatoes, which is caused by an oomycete Phytophthora infestans. The disease is known to cause significant crop losses each year. Many efforts have been made for the effective management of the late blight but the rapid development of new strains of P. infestans is the major constraint. The applicability of host- induced gene silencing (HIGS) in many plants against their pathogen has proven an effective approach and has driven the attention of many researchers. Therefore, in the present study, HIGS approach was adopted to silence the cellulose synthase (CesA) gene(s) of P. infestans to achieve late blight resistance in selected potato cultivars. In the present study, two important potato cvs. ‘Kufri Pukhraj’ and ‘Kufri Chipsona 1’ were selected based on their resistance to late blight disease. The evaluation assay of four Indian potato cultivars (Kufri Pukhraj’, ‘Kufri Chipsona 1’, ‘Kufri Jyoti’ and ‘Kufri Surya’) with the sporal suspension of P. infestans revealed that the cvs. ‘Kufri Pukhraj’ (93.75% DI; 85.41% DS) is susceptible, ‘Kufri Chipsona 1’ (75% DI; 59.83% DS), ‘Kufri Jyoti’ (68.75% DI; 56.25% DS) is moderately resistance and ‘Kufri Surya’ (18.75% DI; 3.46% DS) is resistant to late blight. Therefore, the two cultivars ‘Kufri Pukhraj’ and ‘Kufri Chipsona 1’ were selected for transformation with the Agrobacterium harbouring hp-RNAi constructs specific to the CesA genes of P. infestans. The process of Agrobacterium-mediated genetic transformation requires the optimisation of regeneration protocol as difficulties are faced in shoot organogenesis on selection-cum regeneration medium after co-cultivation. Therefore, in this study the effect of β-lactam antibiotics (used for elimination of Agrobacterium) and sucrose was investigated to improve shoot organogenesis. The effect of β-lactam antibiotics viz. cefotaxime, carbenicillin, and cephalexin was studied on shoot regeneration of potato cvs. ‘Kufri Pukhraj’ and ‘Kufri Chipsona 1’. Leaf and internodal explants from 21-day-old micro shoots were inoculated on MS2 medium (basal MS +10 μM AgNO 3 +10 μM BA+15 μM GA 3 ) further supplemented with different concentrations (0-500 mg L -1 ) of β-lactam antibiotics (cefotaxime, carbenicillin, and cephalexin). These antibiotics promoted shoot organogenesis in both cultivars at lower concentrations, except cephalexin. Cefotaxime (100 mg L -1 ) induced shoot regeneration in maximum explants of cvs. ‘Kufri Pukhraj’ (internode 85.41%; leaf 65.27%) and ‘Kufri Chipsona1’ (75% internodal and 62.5%). Carbenicillin improved shoot regeneration at lower concentrations (< 300 mg L -1 ), whereas cephalexin completely inhibited shoot regeneration in cv. ‘Kufri Chipsona 1’ and

improved in cv. ‘Kufri Pukhraj’ upto 100 mg L -1 . The regeneration efficiency of internodal explants was significantly higher than that of leaf explants. In addition, the effect of sucrose concentration (30-150 mM) was investigated on shoot organogenesis. It was observed that sucrose at 60–90 mM induced higher shoot organogenesis and number of shoots per explant, whereas lower and higher concentrations inhibited the same. Maximum shoot organogenesis was achieved from internodal explants of cv. ‘Kufri Pukhraj’ (84.44%) followed by ‘Kufri Chipsona 1’ (65.55%) on MS3 medium (basal MS +10 μM AgNO 3 +10 μM BA+15 μM GA 3 +100 mg L -1 ) containing 60 mM sucrose. Sucrose was also observed to affects the activity and expression of sucrose metabolising (acid invertase and sucrose synthase) and starch metabolising enzymes (α-amylase and β-amylase). The whole concept of silencing the CesA gene of P. infestans is based upon the differences in sequences at the N-terminal end of PiCesA gene (P. infestans) and StCesA gene (potato). Strategically the ~200 bp non-homologous fragments of PiCesA3 and PiCesA2 gene to that of StCesA genes were selected and amplified in sense and antisense orientation. Two hp-RNAi constructs were developed targeting the PiCesA3 and PiCesA2 genes of P. infestans. Cloning of the ~200 bp selected fragments of PiCesA3 and PiCesA2 gene in sense and antisense orientation along the intron was performed in the hp-RNAi plasmid. The subsequent cloning of both the fragments was confirmed by amplification and restriction digestion of the ~200 bp cloned fragment with specific primers and restriction enzymes respectively. The developed RNAi constructs (PiCesA3-RNAi construct I and PiCesA2-RNAi construct II) were then used to perform the genetic transformation of potato cvs. ‘Kufri Pukhraj’ and ‘Kufri Chipsona 1’. The genetic transformation of the potato cultivars through Agrobacterium needs optimisation of parameters affecting genetic transformation. The highest transient GUS efficiency was recorded with explants precultured for two days and infected with a bacterial density of Agrobacterium (OD 590 ≈0.6) for 15 min and co-cultivated (48 h) on MS4 medium (basal MS +10 μM AgNO 3 +10 μM BA+15 μM GA 3 +100 μM acetosyringone) and screened on MS5 medium (basal MS +10 μM AgNO 3 +10 μM BA+15 μM GA 3 +100 mg L -1 cefotaxime+100 mg L -1 kanamycin). This optimised protocol was used to transform the explants of cv. ‘Kufri Pukhraj’ and ‘Kufri Chipsona 1’. The putatively regenerated transgenic lines were multiplied and maintained on MS1 medium (basal MS +10 μM AgNO 3 ) for further analysis. The PCR analysis of regenerated shoots confirmed the presence of respective sense and antisense fragments, nptII gene, and absence of 16s rRNA gene. The transgenic lines were challenged with P. infestans to evaluate the level of resistance to late blight disease. A significantly lower disease incidence and severity were recorded in

transgenic lines as compared to the control plants. The whole plant assay revealed that in transgenic lines expressing PiCesA3-RNAi construct I, lower disease progression was recorded in line 4 (50% DI; 38.88% DS) of cv. ‘Kufri Pukhraj’ and lines 3 (37.5% DI; 37.49% DS) and line 5 (43.75% DI; 40.97% DS) of cv. ‘Kufri Chipsona 1’ at 10 dpi. However, in transgenic lines expressing PiCesA2-RNAi construct II, minimum disease progression was recorded in line 4 (43.75% DI; 30.5% DS) of cv. ‘Kufri Pukhraj’ and line 1 (43.75% DI; 38.38% DS) and line 7(43.75% DI; 37.49% DS) of cv. ‘Kufri Chipsona 1’ at 10 dpi. The transgenic lines developed using PiCesA2-RNAi construct II showed higher late blight resistance than those developed using PiCesA3-RNAi construct I. Among cultivars, the transgenic lines of cv. ‘Kufri Chipsona 1’ showed higher late blight resistance. Moreover, the activity of four antioxidant enzymes namely superoxide dismutase, catalase, peroxidase, and ascorbate peroxidase, was also studied in inoculated and uninoculated control and transgenic plants. Significant differences in the activities of various enzymes were observed between the inoculated control and transgenic plants. In transgenic lines expressing PiCesA3-RNAi construct I, the maximum activity of superoxide dismutase (12.67 U mg -1 protein), catalase (42.86 U mg -1 protein), peroxidase (85.39 U mg -1 protein), and ascorbate peroxidase (128.17 U mg -1 protein) was recorded in ‘line 4’ of cv. ‘Kufri Pukhraj’, while in cv. ‘Kufri Chipsona 1’ the ‘line 3’ showed the maximum activity of superoxide dismutase (15.96 U mg -1 protein), catalase (56.95 U mg -1 protein), peroxidase (95.33 U mg -1 protein), and ascorbate peroxidase (132.77 U mg -1 protein). In transgenic lines expressing PiCesA2-RNAi construct II, the maximum activity of superoxide dismutase (14.52 U mg -1 protein), catalase (49.45 U mg -1 protein), peroxidase (89.14 U mg -1 protein), and ascorbate peroxidase (125.64 U mg -1 protein), was recorded in ‘line 4’ of cv. ‘Kufri Pukhraj’ while in cv. ‘Kufri Chipsona1’ the ‘line 7’ showed the maximum activity of superoxide dismutase (14.11 U mg -1 protein), catalase (64.02 U mg -1 protein), peroxidase (98.56 U mg -1 protein), and ascorbate peroxidase (134.49 U mg -1 protein).

Keyboards: Solanum tuberosum, Phytophthora infestans, Cellulose synthase gene, Late blight, Host Induced Gene Silencing