RNAi activation with homologous and heterologous sequences that induce resistance against the begomovirus Pepper golden mosaic virus (PepGMV)

Abstract

This study compared the transcriptional changes in Nicotiana benthamiana plants treated with homologous sequences derived from Pepper golden mosaic virus (PepGMV) and heterologous sequences that derived from another begomovirus, Tomato chino La Paz virus (ToChLPV) prior to infection by PepGMV. The results of microarray analyses identified upregulated genes associated with RNAi such as DCL2, DCL4, AGO3, AGO7, AGO10, NRPD2B (Pol IV), DRB3, CMT3, RDR6. The components that participate in different RNAi pathways were identified, including methylation induced by both constructs, as well as the code of these genes in Arabidopsis thaliana and its counterpart in N. benthamiana through different genome assembly. The expression of these genes was validated by quantitative reverse transcription polymerase chain reaction (RT-qPCR), where DCL3, DCL4, AGO1-1, AGO2, RDR6 and PPR1 showed increased expression during plant protection with the heterologous construct compared to those protected with the homologous construct. The results of this study confirmed the activation of the gene silencing mechanism at the transcriptional level with both constructs and established the possibility of their use as a protection system for both homologous and heterologous sequences.

Keywords: Gene silencing; Microarray; Tomato chino La Paz virus (ToChLPV); dsRNA.

Fig. 1

a Graphic results of the evaluation of Z-scores from the microarrays analysis from Nicotiana benthamiana plants protected with CIRP (homologous construct of the intergenic region of PepGMV) or CIRT (heterologous construct of the intergenic region of ToChLPV) and challenged with PepGMV, where ‘up” and “down” values are indicated by green, blue, cyan and white. b Results of the analysis of annotated sequences

Fig. 2

Gene ontology annotation obtained by microarray analysis from Nicotiana benthamiana plants protected with CIRP (homologous construct of the intergenic region of PepGMV) or CIRT (heterologous construct of the intergenic region of ToChLPV) and challenged with PepGMV. Transcripts were grouped into functional Blast2GO groups: biological process, molecular function and cellular component categories with Z-scores 2 and 1.5. The percentages of differentially expressed genes during infection in each category are shown within the parentheses

Fig. 3

STRING network of functional protein associations with gene silencing. a High connectivity is observed among the proteins associated with gene silencing and methylation in CIRT (construct of the intergenic region of ToChLPV), and b subset of proteins involved in gene silencing and methylation in CIRP (construct of the intergenic region of PepGMV). The colors of lines connecting the proteins indicate an interaction

Fig. 4

RT-qPCR expression analysis of DCL2, DCL4, AGO1, AGO2, RDR6 and PPR1 in Nicotiana benthamiana plants protected with CIRP (homologous construct of the intergenic region of PepGMV) or CIRT (heterologous construct of the intergenic region of ToChLPV) and infected with PepGMV. Means indicate significant difference (p < 0.05) among treatments CIRP, CIRT, cv (positive control) and sv (negative control)

Fig. 5

Schematic representation of the proposed model for RNAi-mediated protection of Nicotiana benthamiana against PepGMV infection with CIRP (homologous construct of the intergenic region of PepGMV) and CIRT (heterologous construct of the intergenic region of ToChLPV) based on the results of microarrays and RT-qPCR analysis from the this study and those of several other studies on this topic