Isolation and expression pattern analysis of CmRNF5 and CmNPH3L potentially involved in graft compatibility in cucumber/pumpkin graft combinations

Abstract

Abstract Graft compatibility between the rootstock and scion is a prerequisite for grafting and is presumably regulated by multiple genes involved in a complicated regulatory network. To better understand the mechanism of graft compatibility, it is necessary to analyze the function of differentially expressed genes in graft combinations with different degrees of graft compatibility. The objective of this study was to identify transcriptionally regulated genes and their possible functions in graft compatibility. Using cucumber (Cucumis sativus L.) as scions, pumpkin (Cucurbita L.) with different degrees of graft compatibility as rootstocks, and both self-grafting and non-grafting as controls, 18 different transcript-derived fragments (TDFs) in the leaves of grafted cucumber plants were cloned and sequenced using complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) technique. qRT-PCR showed that 6 TDFs exhibited significant differences between compatible and incompatible grafted cucumber leaves, and two of these TDFs also displayed differences between the two compatible and incompatible rootstocks at 25 days after grafting (DAG). Subsequently, a RING finger protein gene named CmRNF5 and a BTB domain-containing protein gene named CmNPH3L were cloned from pumpkin. Amino acid sequence alignments and phylogenetic analysis showed that these two genes were highly orthologous with those in other plants. The expression patterns of the two genes were investigated in rootstocks with different degrees of compatibility over time (5, 10, 15, 20, and 25 DAG). The results indicated that CmRNF5 and CmNPH3L transcripts were differentially expressed between compatible and incompatible unions throughout graft development. The expression differences of the two genes observed in the scion and different compatibility rootstocks suggested that these two genes could be related to graft compatibility/incompatibility responses. To gain more insight into the regulation of graft development, analysis of the expression patterns of the two genes under different environmental stimuli was performed via qRT-PCR, and the results showed that both genes are stress-induced genes. Based on the bioinformatics analysis of CmRNF5 and CmNPH3L, we hypothesized that CmRNF5 and CmNPH3L may trigger a compatibility/incompatibility response between the two graft partners that constitutes a compatible/incompatible combination via the ABA and auxin signal transduction pathways, respectively.