| Wheat stripe rust,caused by Puccinia striiformis f.sp.tritici（Pst）,is one of the most widely distributed and devastating fungal diseases of wheat in China.For a long time,due to the difficulties of unavailability of culture in vitro and stable genetic transformation of Pst and the large and complex genome of wheat allohexaploid,the research on stripe rust is relatively backward and the disease control is passive.The most cost-effective and efficient approach of prevention and control this disease is rational utilization of new resistant wheat cultivars.However,the traditional rust resistance breeding is facing great challenges due to the frequent variation of Pst virulence and the lack of wheat resistance resources.Therefore,it is urgent to excavate new gene resources for resistance to stripe rust in wheat and carry out research on the mechanism of resistance mediated by disease-resistance factors,which is of great significance for the sustainable green prevention and control of stripe rust in wheat.The resistance response of plants to pathogens involves a series of signal recognition,transduction and expression of defense-related genes.These signaling molecules such as calcium ions(Ca2+),reactive oxygen species（ROS）,salicylic acid（SA）,and brassinosteroid（BR）stimulate the expression of pathogenesis-related genes through complex signaling pathways.However,little work has been reported on molecular mechanisms of wheat broad-spectrum resistance to Pst.In this study,we conducted a series of studies on discovering new disease resistance factors and elucidating their mediated resistance mechanisms and obtained some innovative results as follows.1.TaCSN5 contributes to negative regulation of wheat resistance to Pst in a SA-dependent manner.Previous studies indicated that COP9（constitutional photomorphogenic）signalosome（CSN）plays an important role in plant growth,development,and response to stress conditions.However,the function of wheat CSNs remains elusive between wheat and Pst interaction.With the help of the time series dual RNA-seq data using wheat plants inoculated with Pst revealed that transcript levels of TaCSN5 were induced in compatible interaction.Meanwhile,TaCSN5 was upregulated in response to treatment with SA.So TaCSN5 may be involved in wheat and Pst interaction through SA signaling pathway.To verify its function,TaCSN5 overexpressing Arabidopsis lines were produced,which have significantly increased susceptibility to Pseudomonas syringae pv.tomato DC3000（Pto DC3000）accompanied by down-regulation of At PR1 expression.Further,TaCSN5 overexpression and silencing wheat lines were generated,and TaCSN5 overexpression lines significantly increased susceptibility to Pst accompanied by down-regulation of PR genes expression and increased infection areas.However,TaCSN5 silencing lines showed broad-spectrum resistance to multiple Pst races accompanied by increased transcript levels of PR genes and inhibition of Pst growth.Besides,TaCSN5 negatively regulated the transcription of TaG3NPR1 and accumulation of SA in wheat-Pst interaction.In summary,TaCSN5contributes to negative regulation of wheat resistance to Pst in a SA-dependent manner.2.TaNH2 requires phosphorylation of TaCIPK10 to confer broad-spectrum resistance of wheat to Pst.Previous studies showed that TaCIPK10（CBL（calcineurin B-like）interacting protein kinases 10）phosphorylates Ser（serine）at position 518 of TaNH2（NPR1 homolog）to trigger wheat defense responses to Pst.But the mechanism regulating the response requires further investigation.We generated TaNH2 and TaNH2-S518A（serine（S）→alanine（A）at position518,which can’t be phosphorylated by TaCIPK10）transgenic wheat lines.TaNH2-overexpression（OE）lines expressed a better resistance phenotype than TaNH2-S518A-OE lines,suggesting that TaNH2 requires phosphorylation of TaCIPK10 to confer wheat resistance against Pst.Furthermore,we produced stable TaCIPK10 and TaCIPK10-K42N（lysine（K）→asparagine（N）at position 42,K42N abolishes kinase activity）transgenic wheat lines.Compared with TaCIPK10-OE plants,TaCIPK10-K42N-OE significantly reduced wheat resistance to Pst.However,compared with Fielder plants,phenotypes of TaCIPK10-K42N-OE lines were hardly changed.Thus,the kinase activity of TaCIPK10 is necessary for its function against Pst and TaNH2 requires phosphorylation of TaCIPK10 to confer wheat resistance.3.TaBZR2 confers broad-spectrum resistance to Pst by increasing total chitinase activity in wheat.The brassinosteroid（BR）pathway promotes a variety of physiological processes in plants,and the brassinosteroid insensitive1-ethylmethane sulfonate suppressor（BES）/brassinazole-resistant（BZR）functions as a key transcription factor in the BR signaling process.Previous studies have shown that the BES/BZR transcription factor TaBZR2 mediates drought stress response in wheat（Triticum aestivum）by directly activating glutathione S-transferase 1（TaGST1）transcription.However,the function of TaBZR2 under biotic stresses is unknown.With the help of the time series dual RNA-seq data using wheat plants inoculated with Pst revealed that transcript levels of TaBZR2 were induced.To investigate whether TaBZR2 is involved in wheat immunity,the expression analysis revealed that the transcript levels of TaBZR2 were significantly up-regulated after inoculation with Pst and treatment with flg22 or Pst322（an elicitor-like protein of Pst）.To further verify its function,TaBZR2 overexpression and silencing wheat lines were generated,and TaBZR2 overexpression lines conferred resistance to multiple Pst races accompanied by reduced fungal growth,increased transcript levels of PR genes,and increased H2O2accumulation near the infection site,whereas TaBZR2 silencing lines showed increased susceptibility to multiple Pst races accompanied by increased infection area,decreased transcript levels of PR genes,and decreased H2O2 accumulation near the infection sites.Further,transient silencing of TaGST1 in wheat revealed no significant difference in phenotype compared with the control during wheat-Pst interaction,which suggested that TaGST1,which is activated by TaBZR2 under drought,doesn’t play a major role in Pst infection.To further explore the disease-resistance mechanism,transcriptome analysis was performed on TaBZR2-OE lines during wheat-Pst interaction.The Go（gene ontology）analysis indicated that TaBZR2 may regulate transcription of wheat chitinase genes.Electrophoretic mobility shift assay（EMSA）and dual-luciferase reporter gene assay indicated that TaBZR2 targeted the promoter of the chitinase gene TaCht20.2 to activate its transcription.Chitinase activity assay showed that TaBZR2 increased chitinase activity in wheat challenged by Pst infection.In summary,TaBZR2 conferred broad-spectrum resistance to Pst by increasing total chitinase activity in wheat.In summary,the present study systematically characterized the functions of TaCSN5,TaNH2,and TaBZR2 during wheat-Pst interaction,dissected the TaBZR2-mediated mechanism,enriched plant immune system network mediated by SA and BR signaling pathways,and provided promising genetic resources and theoretical basis for wheat rust resistance breeding. |
