Functional And Mechanism Analysis Of Wheat Genes TaRCR1 And TaAGC1 In Defense Response To Sharp Eyespot

Recently with the changes of climate, farming system and planting density, wheat sharp eyespot caused Rhizoctonia cerealis, has become one of the destructive diseases of wheat in China. More than one hundred million acres of wheat has been infected by R. cerealis since 2005, which caused serious economic losses. The genetics of wheat resistance to sharp eyespot is not fully understood, and progress of the corresponding traditional breeding is slowly, so it has important significance to explore defense-related genes and unravel the wheat defense mechanisms against R. cerealis. NB-LRR protein and protein kinase are two important proteins in plant defense response. In this study, TaRCR1-overexpressing and-silencing, TaAGC1-overexpressing and-silencing wheat plants were used as experimental materials to study the function and mechanism of TaRCR1 and TaAGC1 whose expression was induced with R. cerealis.TaRCR1 encodes NB-LRR type protein which involves in the defense response of wheat to sharp eyespot. In this study, the molecular characteristics, function, and mechanism of TaRCR1 were studied, and the main results are as follows:（1） The expression of TaRCR1 was induced with R. cerealis inoculation and the induction reached a peak at 7 dpi, and the expression level in the roots and stems were more abundant than those in the leaves and spikes.（2） Transient expression of TaRCR1 in wheat protoplast indicated that the TaRCR1 protein was localized to the cytoplasm and nucleus.（3） Molecular characteristics of TaRCR1 were analyzed by PCR, qRT-PCR, and western blotting in T3-T5 generation of TaRCR1 transgenic wheat. The results showed that the TaRCR1 gene can inherit, transcript, and overexpress in six transgenic lines. The Investigation on the resistance of TaRCR1 transformed Yangmai 16 to sharp eyespot showed that TaRCR1 overexpression enhanced resistance of wheat to sharp eyespot.（4） Barley stripe mosaic virus-induced gene silencing（BSMV-VIGS） was used to silence the expression of TaRCR1 in CI12633 plant. After infection with R. cerealis for 12 d, compared with mock plants（BSMV:00-infected plants）, the TaRCR1-silencing plants（BSMV:TaRCR1-infected plants） showed more more obvious symptoms of sharp eyespot, suggesting that silence of TaRCR1 compromised wheat resistance to R. cerealis.（5） The expression patterns of 14 defense-related genes in TaRCR1-overexpressing lines, wild-type（WT） Yangmai 16 and TaRCR1-silencing plants and BSMV:00-infected controls were analyzed by qRT-PCR. The results showed that TaRCR1 positively regulated the expression of PR1, PR2, chitinase2, and TaPIE1, which was induced by R. cerealis stress.（6） TaRCR1 was highly induced at early stage of H₂O₂ and JA stimulus, and the induced transcriptional abundance of TaRCR1 by R. cerealis infection was significantly elevated following by H₂O₂ pretreatment.（7） The peroxidase（POD） activity and transcriptional levels of two ROS-scavenging enzyme genes CAT1 and POX2 were up-regulated, and that of NOX encoding ROS-producing enzyme was down-regulated by TaRCR1. The results of DAB and NBT staining showed that TaAGC1 overexpression reduced the overproduction of ROS in the transgenic wheat induced by R. cerealis inoculation. Following the treatment with POD inhibitor NaN3, the difference in R. cerealis resistance between the TaRCR1 transgenic and WT plant was largely diminished. These results suggested ROS-homeostasis plays an important role in TaRCR1-mediated resistance to R. cerealis, and TaRCR1 can maintain ROS-homeostasis through regulating expression of ROS-related genes, finally leading to enhanced resistance to R. cerealis.（8） The total protein of TaRCR1-overexpressing plants was extracted and purified. After digested by trypsase, mass spectrometric analysis was performed to identify protein interacting with TaRCR1. A kinase named TaPP2 Ac was identified and was proved to interact with TaRCR1 by yeast two-hybrid. The BSMV-VIGS experiment showed that silence of TaPP2 Ac enhanced wheat resistance to R. cerealis. And the result of qRT-PCR analysis showed that the transcriptional level of TaPP2 Ac in TaRCR1-overexpressing plants was lower than that in WT plants and was highest in the TaRCR1-silence plants. These results suggest that TaRCR1 may elevated the wheat resistance to R. cerealis through negatively regulated the expression of TaPP2 Ac gene.Our previous study showed that the overexpression of TaAGC1 increased the resistance level of wheat to sharp eyespot, while silence of TaAGC1 decreased the level. In this paper, the mechanism of TaAGC1 mediated resistance to R. cerealis was studied, and the main results are as follows:（1） TaAGC1 is responsive to H₂O₂ stimulate, which induced the expression of TaAGC1, and reached the peak at 30 min post H₂O₂ treatment, suggesting that TaAGC1 might be involve in H₂O₂ signal pathway.（2） The results of DAB and NBT staining showed that the R. cerealis infection promoted the generation of reactive oxygen species（ROS, like H₂O₂ and O2-）, and TaAGC1 overexpression reduced the accumulation of ROS in the transgenic wheat. The transcription of wheat genes encoding ROS-scavenging enzymes（POX2, CAT1 and SOD3） and ROS-producing enzyme（NOX） were investigated by qRT-PCR, and the results showed that expression levels of POX2, CAT1 and SOD3 genes were significantly higher in TaAGC1 overexpressing wheat lines than in the WT plants and were lowest in the TaAGC1 silence plants. However, NOX transcript level was significantly lower in TaAGC1-overexpressing lines than in WT plants and was the highest in TaAGC1-silencing plants.（3） qRT-PCR analysis showed that the expression of four wheat defence-associated genes, defensin, nsLTP1, PR10 and chitinase2, was significantly induced in WT wheat after R. cerealis inoculation for 7 d, 14 d or 40 d. TaAGC1 positively regulated the transcriptional levels of wheat defensin and nsLTP1 genes, while negatively regulated those of PR10 and chitinase2. In summary, TaAGC1, acting as a positive regulator, mediates host resistance to sharp eyeapot through modulating the expression of ROS-related and certain defence-associated genes.