Abstract Although cytosolic free Ca2+ mobilization induced by microbe/pathogen-associated molecular patterns is postulated to play a pivotal role in innate immunity in plants, the molecular links between Ca2+ and downstream defense responses still remain largely unknown. Calcineurin B-like proteins (CBLs) act as Ca2+ sensors to activate specific protein kinases, CBL-interacting protein kinases (CIPKs). We here identified two CIPKs, OsCIPK14 and OsCIPK15, rapidly induced by microbe-associated molecular patterns, including chitooligosaccharides and xylanase (Trichoderma viride/ethylene-inducing xylanase [TvX/EIX]), in rice (Oryza sativa). Although they are located on different chromosomes, they have over 95% nucleotide sequence identity, including the surrounding genomic region, suggesting that they are duplicated genes. OsCIPK14/15 interacted with several OsCBLs through the FISL/NAF motif in yeast cells and showed the strongest interaction with OsCBL4. The recombinant OsCIPK14/15 proteins showed Mn2+-dependent protein kinase activity, which was enhanced both by deletion of their FISL/NAF motifs and by combination with OsCBL4. OsCIPK14/15-RNAi transgenic cell lines showed reduced sensitivity to TvX/EIX for the induction of a wide range of defense responses, including hypersensitive cell death, mitochondrial dysfunction, phytoalexin biosynthesis, and pathogenesis-related gene expression. On the other hand, TvX/EIX-induced cell death was enhanced in OsCIPK15-overexpressing lines. Our results suggest that OsCIPK14/15 play a crucial role in the microbe-associated molecular pattern-induced defense signaling pathway in rice cultured cells.
Signal molecules derived from pathogens/microbes or plants (pathogen/microbe/damage-associated molecular patterns; PAMPs/MAMPs/DAMPs), elicitors, trigger changes in cytosolic free Ca2+ concentrations ([Ca2+]cyt) to activate plant immune responses. A rice two-pore channel 1 (OsTPC1) has been suggested to be involved in fungal xylanase elicitor (TvX)-induced defense responses including [Ca2+]cyt increase, phytoalexin production and hypersensitive cell death in suspension-cultured rice cells. However, little is known on the molecular links between [Ca2+]cyt rise and elicitor-induced gene expression. To gain insights on the possible roles of OsTPC1 in TvX-induced gene expression, we performed DNA microarray analysis using a rice 44K oligo-microarray system, and revealed that TvX induce expression of thousands of genes including WRKY-type transcription factors, a serine hydrolase involved in hypersensitive cell death, and diterpene cyclases required for phytoalexin biosynthesis, which are suppressed in the Ostpc1 knockout mutant. TvX-induced expression of genes involved in the methylerythritol phosphate (MEP) pathway, which is located upstream of the phytoalexin biosynthesis pathway, was also suppressed in Ostpc1 cells. Possible involvement of OsTPC1 in the regulation of gene expression and metabolism in cultured-rice cells is discussed.
