Plant and animal perception of microbes through pathogen surveillance proteins leads to MAP kinase signalling and the expression of defence genes. However, little is known about how plant MAP kinases regulate specific gene expression. We report that, in the absence of pathogens, Arabidopsis MAP kinase 4 (MPK4) exists in nuclear complexes with the WRKY33 transcription factor. This complex depends on the MPK4 substrate MKS1. Challenge with Pseudomonas syringae or flagellin leads to the activation of MPK4 and phosphorylation of MKS1. Subsequently, complexes with MKS1 and WRKY33 are released from MPK4, and WRKY33 targets the promoter of PHYTOALEXIN DEFICIENT3 (PAD3) encoding an enzyme required for the synthesis of antimicrobial camalexin. Hence, wrky33 mutants are impaired in the accumulation of PAD3 mRNA and camalexin production upon infection. That WRKY33 is an effector of MPK4 is further supported by the suppression of PAD3 expression in mpk4-wrky33 double mutant backgrounds. Our data establish direct links between MPK4 and innate immunity and provide an example of how a plant MAP kinase can regulate gene expression by releasing transcription factors in the nucleus upon activation.

The HMG-box domain of approximately 75 amino acid residues was originally identified as the domain that mediates the DNA-binding of chromatin-associated high-mobility group (HMG) proteins of the HMGB type. In the last few years, HMG-box domains have been found in various DNA-binding proteins including transcription factors and subunits of chromatin-remodeling complexes. HMG-box domains mediate either non-sequence-specific (e.g., HMGB-type proteins) or sequence-specific (e.g., transcription factors) DNA binding. Both types of HMG-box domains bind non-B-type DNA structures (bent, kinked and unwound) with high affinity. In addition, HMG-box domains are involved in a variety of protein-protein interactions. Here, we have examined the human and plant genomes for genes encoding HMG-box domains. Compared to plants, human cells contain a larger variety of HMG-box proteins. Whereas in humans transcription factors are the most divergent group of HMG-box proteins, in plants the chromosomal HMGB-type proteins are most variable.

HMG1 has two homologous, folded DNA-binding domains ("HMG boxes"), A and B, linked by a short basic region to an acidic C-terminal domain. Like the whole protein, which may perform an architectural role in chromatin, the individual boxes bind to DNA without sequence specificity, have a preference for distorted or prebent DNA, and are able to bend DNA and constrain negative superhelical turns. They show qualitatively similar properties with quantitative differences. We have previously determined the structure of the HMG box from the central B-domain (77 residues) by two-dimensional NMR spectroscopy, which showed that it contains a novel fold [Weir et al. (1993) EMBO J. 12, 1311-1319]. We have now determined the structure of the A-domain (as a Cys-->Ser mutant at position 22 to avoid oxidation, without effect on its DNA-binding properties or structure) using heteronuclear three- and four-dimensional NMR spectroscopy. The A-domain has a very similar global fold to the B-domain and the Drosophila protein HMG-D [Jones et al. (1994) Structure 2, 609-627]. There are small differences between A and B, in particular in the orientation of helix I, where the B-domain is more similar to HMG-D than it is to the A-domain; these differences may turn out to be related to the subtle differences in functional properties between the two domains [Teo et al. (1995) Eur. J. Biochem. 230, 943-950] and will be the subject of further investigation. NMR studies of the interaction of the A-domain of HMG1 with a short double-stranded oligonucleotide support the notion that the protein binds via the concave face of the L-shaped structure; extensive contacts with the DNA are made by the N-terminal extended strand, the N-terminus of helix I, and the C-terminus of helix II. These contacts are very similar to those seen in the LEF-1 and SRY-DNA complexes [Love et al. (1995) Nature 376, 791-795; Werner et al. (1995) Cell 81, 705-714].

Transcript elongation factors (TEFs) are a heterogeneous group of proteins that control the efficiency of transcript elongation of subsets of genes by RNA polymerase II (RNAPII) in the chromatin context. Using reciprocal tagging in combination with affinity purification and mass spectrometry, we demonstrate that in Arabidopsis thaliana, the TEFs SPT4/SPT5, SPT6, FACT, PAF1-C, and TFIIS copurified with each other and with elongating RNAPII, while P-TEFb was not among the interactors. Additionally, NAP1 histone chaperones, ATP-dependent chromatin remodeling factors, and some histone-modifying enzymes including Elongator were repeatedly found associated with TEFs. Analysis of double mutant plants defective in different combinations of TEFs revealed genetic interactions between genes encoding subunits of PAF1-C, FACT, and TFIIS, resulting in synergistic/epistatic effects on plant growth/development. Analysis of subnuclear localization, gene expression, and chromatin association did not provide evidence for an involvement of the TEFs in transcription by RNAPI (or RNAPIII). Proteomics analyses also revealed multiple interactions between the transcript elongation complex and factors involved in mRNA splicing and polyadenylation, including an association of PAF1-C with the polyadenylation factor CstF. Therefore, the RNAPII transcript elongation complex represents a platform for interactions among different TEFs, as well as for coordinating ongoing transcription with mRNA processing.

SUMMARYThe facilitates chromatin transcription (FACT) complex, consisting of the SSRP1 and SPT16 proteins, is a histone chaperone that assists the progression of transcribing RNA polymerase on chromatin templates by destabilizing nucleosomes. Here, we examined plants that harbour mutations in the genes encoding the subunits of Arabidopsis FACT. These experiments revealed that (i) SSRP1 is critical for plant viability, and (ii) plants with reduced amounts of SSRP1 and SPT16 display various defects in vegetative and reproductive development. Thus, mutant plants display an increased number of leaves and inflorescences, show early bolting, have abnormal flower and leaf architecture, and their seed production is severely affected. The early flowering of the mutant plants is associated with reduced expression of the floral repressor FLC in ssrp1 and spt16 plants. Compared to control plants, reduced amounts of FACT in mutant plants are detected at the FLC locus as well as at the locations of housekeeping genes (whose expression is not affected in the mutants), suggesting that expression of FLC is particularly sensitive to reduced FACT activity. Analysis of double mutants that are affected in the expression of both FACT subunits and factors catalysing the mono-ubiquitination of histone H2B (HUB1/2) demonstrates that they genetically interact to regulate various developmental processes (i.e. branching, leaf venation pattern, silique development) but independently regulate the growth of leaves and the induction of flowering.