| Plant resistances to pathogens are achieved either through cell-surface pattern-recognition receptors (PRRs) that recognize microbe-associated molecular patterns (MAMPs) or via intracellular nucleotide-binding leucine-rich repeat (NB-LRR) immune sensors as effector proteins. These processes initiate overlapping and distinct signaling pathways involving protein kinase activation, Ca2+influx, hormone biosynthesis, oxidative burst and transcriptional reprogramming. Among them, Ca2+plays an important role as a secondary messenger which can be sensed by calcium-dependent protein kinases (CDPKs, or lately named CPKs). Wheat is one of the most important staple crops of mankind, in which biotic and abiotic stresses are major factors affecting its growth and productivity. CPKs are important Ca2+signaling components involved in complex immune and stress signaling networks, but the knowledge of CPK gene functions in the hexaploid wheat is limited. Previously, we showed that TaCPK2was inducible by powdery mildew (PM; Blumeria graminis tritici, Bgt) infection in wheat. Here we further characterized TaCPK2-A functions in disease resistance.Using a previously reported sequence as a template for primer design (AY704444), TaCPK2cDNA sequences were obtained from the hexaploid Chinese Spring (CS) and distinguished them according to their polymorphisms with diploid cDNA sequences. The three cDNAs were named TaCPK2-A, TaCPK2-B, and TaCPK2-D derived from A, B, and D subgenomes respectively. The three homoeologous genes contained open reading frames of1,677bp,1,680bp and1,671bp long that encoded558,559and556amino acid (aa) respectively. The three proteins were highly similar with96.9-98.6%identity. Compared with their rice ortholog OsCPK13, most divergent amino acids of wheat CPK13proteins were located at the N-terminal domain of the protein whereas key amino acids LGQGQFGT, K and D in the kinase domain and four EF hands (key amino acids D, D, S, E and D, D, D, E) were conserved, indicating that all three homoeologous genes should encode functional CPK proteins.Based on the SNPs at the N-terminal variable region, we designed gene specific primer. The chromosome assignments of the TaCPK2-A, TaCPK2-B, and TaCPK2-D genes were determined using the CS nulli-tetrasomic (NT) lines. The results showed that TaCPK2-A, TaCPK2-B and TaCPK2-D are specific mapped to chromosomes2A,2B, and2D, respectively.Taking advantage of recently published A (T. uratu) and D (Ae. tauschii) draft genome sequences, we isolated two fragments corresponding to1.5kb up-stream the start codon of TaCPK2-A and TaCPK2-D in CS. Cis-element prediction using the program PLACE (http://www.dna.affrc.go.jp/PLACE/signalscan.html) showed two defense responsive WBOXATNPR1(WBO) elements at the promoter region of TaCPK2-A gene. In contrast, two cold responsive LTRECOREATCOR15(LTR) elements were predicted on the TaCPK2-D promoter. To confirm this, we performed quantitative real time PCR (qRT-PCR) using gene specific primers where TaCPK2-A responded to Bgt treatment only while the expression of TaCPK2-D responded weakly to Bgt treatment, but strongly to cold, suggesting that the divergence of these two promoters may lead to subfunctionalization of the two genes.Down regulation of TaCPK2-A by virus-induced gene silencing (VIGS) in leaves of PmA, a resistant wheat line, promoted the compatible interaction between Bgt and PmA, indicating that TaCPK2-A is indeed required for PM resistance. qRT-PCR showed that TaCPK2-A was significantly repressed in VIGS plants. To further confirm that down regulation of TaCPK2-A indeed enhanced Bgt penetration and growth, we performed microscopic examinations on leaves spread with Bgt spores. The results showed that there were more Bgt hyphae on BSMV:TaCPK2-A leaves than the BSMV:GFP control, indicating that the TaCPK2-A VIGS plants were more susceptible to PM infection. Statistic analysis showed significant difference between PM penetration efficiencies (PE) of VIGS plants and the control (19.01±6.13%vs0%). Together, these data suggest that maintaining an overall high level of TaCPK2-A transcripts is essential for PM resistance in wheat.Over expressing TaCPK2-A in Kuiku131, a Xoo susceptible japonica rice cultivar, enhanced its resistance to bacterial blight for both seedling and booting stage plants. The lesion areas on Xoo inoculated leaves were reduced to6.78%,5.06%, and4.32%for transgenic plants line1, line2, and line3respectively, when compared with the control, which is correlated with TaCPK2-A transgene expression levels. To further confirm that only TaCPK2-A transcripts was over expressed and the rice ortholog was not affected, we detected the expression levels of OsCPK13and found that it was indeed not affected in TaCPK2-A transgenic plants. Together, these data indicate that the enhanced Xoo resistance was indeed caused by the increased expression of TaCPK2-A.To ascertain that defense-responsive genes were inducible by TaCPK2-A, we analyzed the expression of OsWRKY45-1as well as10down stream genes in the transgenic rice plants, most of which were SA or JA signaling genes. In Kuiku131transgenic, over expression of TaCPK2-A activated the expressions of WRKY45-1, LOX, AOS2, PR1α and PR1b(P<0.05) upon Xoo infection compared with the wild type,. The expression of SA related genes such as WRKY13, PAL, ICS1, PAD4, NH1, and PR10however were not significantly affected. These results suggest that TaCPK2-A may play an important role in regulating OsWRKY45-1and JA signaling pathway related genes. By contrast, in wheat leaves where TaCPK2-A was repressed by VIGS, the homolog (Ta-05961) of rice OsWRKY45-1was significantly down regulated (P<0.05) when compared with the control BSMV:GFP plants. Correspondingly, LOX (Ta-2498165446) and AOS2(Ta-134687) were down regulated, together with two canonical PR genes PR1α (Ta-01596) and PR1b (Ta-141027), suggesting a similar regulatory mechanism for TaCPK2-A in wheat. In wheat, over expression of TaWRKY45confers disease resistance to multiple fungal diseases in transgenic wheat plants, reinforcing the idea that TaCPK2-A play a role in fungal resistance in wheat via its regulation on TaWRKY45. Together, our data indicate conserved mechanisms mediated by TaCPK2-A for PM (a fungus) resistance in wheat and bacterial blight (a bacterium) resistance in rice. In a word, our data propose that modulation of WRKY45-1and associated defense responsive genes by CPK2genes may be the common mechanism for multiple disease resistances in grass species, which may have undergone subfunctionalization in promoters before the formation of hexaploid wheat. |
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