CabZIP23 Integrates in CabZIP63–CaWRKY40 Cascade and Turns CabZIP63 on Mounting Pepper Immunity against Ralstonia solanacearum via Physical Interaction

Abstract

CabZIP63 and CaWRKY40 were previously found to be shared in the pepper defense response to high temperature stress (HTS) and to Ralstonia solanacearum inoculation (RSI), forming a transcriptional cascade. However, how they activate the two distinct defense responses is not fully understood. Herein, using a revised genetic approach, we functionally characterized CabZIP23 in the CabZIP63–CaWRKY40 cascade and its context specific pepper immunity activation against RSI by interaction with CabZIP63. CabZIP23 was originally found by immunoprecipitation-mass spectrometry to be an interacting protein of CabZIP63-GFP; it was upregulated by RSI and acted positively in pepper immunity against RSI by virus induced gene silencing in pepper plants, and transient overexpression in Nicotiana benthamiana plants. By chromatin immunoprecipitation (ChIP)-qPCR and electrophoresis mobility shift assay (EMSA), CabZIP23 was found to be directly regulated by CaWRKY40, and CabZIP63 was directly regulated by CabZIP23, forming a positive feedback loop. CabZIP23–CabZIP63 interaction was confirmed by co-immunoprecipitation (CoIP) and bimolecular fluorescent complimentary (BiFC) assays, which promoted CabZIP63 binding immunity related target genes, including CaPR1, CaNPR1 and CaWRKY40, thereby enhancing pepper immunity against RSI, but not affecting the expression of thermotolerance related CaHSP24. All these data appear to show that CabZIP23 integrates in the CabZIP63–CaWRKY40 cascade and the context specifically turns it on mounting pepper immunity against RSI.

CaSTH2 disables CaWRKY40 from activating pepper thermotolerance and immunity against Ralstonia solanacearum via physical interaction

“…By previous studies, CaWRKY40 is promoted to activate PR genes such as CaPR4 , CaPR1 , CaDEF1 , and CaHSP24 by CaWRKY27b and CaWRKY28 [ 23 , 58 ], and CaWRKY40 is repressed by CaSTH2 to activate the above-mentioned target genes not by repressing the direct targeting of these genes by CaWRKY40; we speculate that CaSTH2 might repress CaWRKY40 by blocking its interaction with CaWRKY27b or CaWRKY28. We used the BiFC assay to verify this assumption, and the results showed that the co-transient overexpression of CaSTH2 significantly reduced the CaWRKY40-CaWRKY27b/CaWRKY28 interaction ( Fig.…”

“…2C ), lower level of bacterial growth at 48 and 72 hpi ( Fig. 2D ), as well as upregulation of immunity-related CaPR1 , CaPR4 , and CaDEF1 [ 23 ] in contrast to plants of the wild type ( Fig. 2E ).…”

“…The involvement of CaSTH2 and CaWRKY40 in both pepper thermotolerance and in immunity against RSI and their interaction imply that the role that CaWRKY40 plays might be modified by CaSTH2 via physical interaction. To test this speculation, we first performed electrophoretic mobility shift assay (EMSA) using biotin labeled W-box with promoter segments of CaPR1 , CaDEF1 , CaHSP24 , and CaHSFB2a , which are targeted by CaWRKY40 [ 23 , 64 ], and prokaryotic expressed CaSTH2–6 × His and CaWRKY40–6 × His. The result showed that CaWRKY40 bound to all of the tested promoter fragments, and the addition of excess CaSTH2 did not affect these bindings ( Fig.…”

“…According to earlier research, CaWRKY40 activates PR or HSP genes and forms transcriptional cascades with CaWRKY6 [ 39 ], CaWRKY22 [ 59 ], CaWRKY30 [ 60 ], CabZIP23 [ 23 ], and CabZIP63 [ 22 ] to form positive feedback loops that act as protective buffers against RSI or thermotolerance. Additionally, CaWRKY40’s transcriptional activations of target genes related to immunity or thermotolerance are facilitated by CaWRKY27b [ 41 ] or CaWRKY28 [ 42 ].…”

“…We previously found that CaWRKY40 acts positively not only in pepper immunity against RSI but also in defense response to HTS by targeting various immunity or thermotolerance-related genes via W-box in their promoter as well as by autoregulation at transcription level by the DWE, in its own promoter [ 58 ]. Upon RSI or HTS exposure, CaWRKY40 forms transcription cascades with TFs such as CaWRKY6, CaWRKY22, CabZIP23, and CabZIP63 [ 22 , 23 , 39 , 59–61 ] as well as Ca 2+ sensor CaCDPK15 [ 62 ], and is promoted by CaWRKY27b in pepper immunity against RSI and thermotolerance but by CaWRKY28 only in pepper immunity against RSI via physical interaction [ 41 , 42 ]. In this way, CaWRKY40 forms positive feedback loops with WRKY TFs or other TFs to rapidly and efficiently activate immunity or thermotolerance upon perception of the stress.…”