部分敲除NtPDK1a/1b/1c/1d增强烟草抗病性

Partial knockout of NtPDK1a/1b/1c/1d enhances the disease resistance of Nicotiana tabacum

摘要:

真核细胞AGC蛋白激酶家族(AGC kinase family)参与调控多种生物学过程。3-磷酸肌醇依赖性蛋白激酶1(3-phosphoinositide-dependent protein kinase 1,PDK1)是一种保守的丝氨酸/苏氨酸激酶。为了研究PDK1同源基因在四倍体烟草免疫中的作用，对前期获得的同时敲除4个NtPDK1同源基因中5–7个等位基因的CRISPR/Cas9转基因株系进行了细胞死亡及抗病性分析，结果发现，部分敲除NtPDK1a/1b/1c/1d虽然可延迟瞬时过表达激活态Pto (active Pto,Pto^Y207D)及大豆GmMEKK1诱导的超敏反应(hypersensitive response,HR)细胞死亡，但可显著增强烟草对假单胞杆菌(Pseudomonas syrangaepv. tomato DC3000,Pst DC3000)以及烟草花叶病毒(tobacco mosaic virus,TMV)的抗性，而该抗性的增强与NtPDK1a/1b/1c/1d部分敲除株系中NtMPK6、NtMPK3和NtMPK4激活程度的显著增强相关联。综上所述，本研究结果表明NtPDK1a/1b/1c/1d正向调控细胞死亡，但却可能通过抑制MAPK途径的激活而负向调控烟草免疫反应。

Abstract:

The protein kinase A/protein kinase G/protein kinase C-family (AGC kinase family) of eukaryotes is involved in regulating numerous biological processes. The 3-phosphoinositide- dependent protein kinase 1 (PDK1), is a conserved serine/threonine kinase in eukaryotes. To understand the roles of PDK1 homologous genes in cell death and immunity in tetraploid Nicotiana tabacum, the previuosly generated transgenic CRISPR/Cas9 lines, in which 5–7 alleles of the 4 homologous PDK1 genes (NtPDK1a/1b/1c/1d homologs) simultaneously knocked out, were used in this study. Our results showed that the hypersensitive response (HR) triggered by transient overexpression of active Pto (Pto^Y207D) or soybean GmMEKK1 was significantly delayed, whereas the resistance to Pseudomonas syrangae pv. tomato DC3000 (Pst DC3000) and tobacco mosaic virus (TMV) was significantly elevated in these partial knockout lines. The elevated resistance to Pst DC3000 and TMV was correlated with the elevated activation of NtMPK6, NtMPK3, and NtMPK4. Taken together, our results indicated that NtPDK1s play a positive role in cell death but a positive role in disease resistance, likely through negative regulation of the MAPK signaling cascade.