1. ¹College of Biological Sciences, China Agricultural University, Beijing 100094, China;
2. ²National Institute of Biological Science, Zhongguancun Life Science Park, 7 Science Park Road, Beijing 102206, China;
3. ³State Key Laboratories for AgroBiotechnology, China Agricultural University, Beijing 100094, China

Correspondence: Yuelin Zhang, Tel: +86-010-80726688-8360; Fax: +86-010-80728646
E-mail: zhangyuelin@nibs.ac.cn

^*These two authors contributed equally to this work.

Received 24 July 2008; Revised 1 September 2008; Accepted 10 September 2008.

Abstract

Mitogen-activated protein kinase (MAPK) cascades play important roles in regulating plant innate immune responses. In a genetic screen to search for mutants with constitutive defense responses, we identified multiple alleles of mpk4 and mekk1 that exhibit cell death and constitutive defense responses. Bimolecular fluorescence complementation (BiFC) analysis showed that both MPK4 and MEKK1 interact with MKK1 and MKK2, two closely related MAPK kinases. mkk1 and mkk2 single mutant plants do not have obvious mutant phenotypes. To test whether MKK1 and MKK2 function redundantly, mkk1 mkk2 double mutants were generated. The mkk1 mkk2 double mutant plants die at seedling stage and the seedling-lethality phenotype is temperature-dependent. Similar to the mpk4 and mekk1 mutants, the mkk1 mkk2 double mutant seedlings accumulate high levels of H₂O₂, display spontaneous cell death, constitutively express Pathogenesis Related (PR) genes and exhibit pathogen resistance. In addition, activation of MPK4 by flg22 is impaired in the mkk1 mkk2 double mutants, suggesting that MKK1 and MKK2 function together with MPK4 and MEKK1 in a MAP kinase cascade to negatively regulate innate immune responses in plants.