The hypersensitive response (HR) is one of the most critical defense systems in higher plants. In order to understand its molecular basis, we have screened tobacco genes that are transcriptionally activated during the early stage of the HR by the differential display method. Among six genes initially identified, one was found encoding a 57 kDa polypeptide with 497 amino acids not showing significant similarity to any reported proteins except for the AAA domain (ATPase associated with various cellular activities) spanning over 230 amino acids. The bacterially expressed protein exhibited ATP hydrolysis activity, and a green fluorescent protein-fusion protein localized in the cytoplasm of onion epidermis cells. The protein was subsequently designated as NtAAA1 (Nicotiana tabacum AAA1). NtAAA1 transcripts were induced 6 h after HR onset not only by TMV but also by incompatible Psuedomonas syringae, indicating that NtAAA1 is under the control of the N-gene with a common role in pathogen responses. Expression of NtAAA1 was induced by jasmonic acid and ethylene, but not by salicylic acid (SA). It also occurred at a high level in SA-deficient tobacco plants upon TMV infection. When NtAAA1 was silenced by the RNAi method, accumulation of transcripts for PR-1a significantly increased during the HR. Treatments with SA induced higher expression of PR-1a and acidic PR-2 in RNAi transgenic plants than in wild-type counterparts. These results suggest that NtAAA1 mitigates the SA signaling pathway, and therefore that NtAAA1 modulates the pathogen response of the host plants by adjusting the HR to an appropriate level.
Wound-induced protein kinase (WIPK) is a tobacco (Nicotiana tabacum) mitogen-activated protein kinase known to play an essential role in defense against wounding and pathogens, although its downstream targets have yet to be clarified. This study identified a gene encoding a protein of 648 amino acids, which directly interacts with WIPK, designated as N. tabacum WIPK-interacting factor (NtWIF). The N-terminal region with approximately 250 amino acids showed a high similarity to the plant-specific DNA binding domain, B3, but no other similarity with known proteins. The C terminus of approximately 200 amino acids appeared to be essential for the interaction with WIPK, and a Luciferase-reporter gene assay using Bright Yellow 2 cells indicated the full-length protein to possess trans-activation activity, located to the middle region of approximately 200 amino acids. In vitro phosphorylation assays indicated that WIPK efficiently phosphorylates the full-length protein and the N terminus but not the C terminus. When full-length NtWIF was coexpressed with WIPK in Bright Yellow 2 cells, the Luciferase transcriptional activity increased up to 5-fold that of NtWIF alone, whereas no effect was observed with a kinase-deficient WIPK mutant. Transcripts of NtWIF began to simultaneously accumulate with those of WIPK 30 min after wounding and 1 h after the onset of hypersensitive response upon tobacco mosaic virus infection. These results suggest that NtWIF is a transcription factor that is directly phosphorylated by WIPK, thereby being activated for transcription of target gene(s) involved in wound and pathogen responses.
NtWIF is a transcription factor activated upon phosphorylation by wound-induced protein kinase (WIPK) in tobacco plants. Transgenic tobacco plants overexpressing NtWIF exhibited constitutive accumulation of transcripts for pathogenesis-related genes, PR-1a and PR-2. Salicylic acid levels were 50-fold higher than those in wild-type plants. The levels of jasmonic acid and IAA did not significantly differ, while an increase of ABA upon wounding was delayed by 3 h in the transgenics. When challenged with tobacco mosaic virus, lesions developed faster and were smaller in the transgenic plants. The results suggest that NtWIF is likely to influence salicylic acid biosynthesis, being located downstream of WIPK.
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