MEKK1 is required for flg22-induced MPK4 activation in Arabidopsis plants.

The Arabidopsis (Arabidopsis thaliana) gene MEKK1 encodes a mitogen-activated protein kinase kinase kinase that has been implicated in the activation of the map kinases MPK3 and MPK6 in response to the flagellin elicitor peptide flg22. In this study, analysis of plants carrying T-DNA knockout alleles indicated that MEKK1 is required for flg22-induced activation of MPK4 but not MPK3 or MPK6. Experiments performed using a kinase-impaired version of MEKK1 (K361M) showed that the kinase activity of MEKK1 may not be required for flg22-induced MPK4 activation or for other macroscopic FLS2-mediated responses. MEKK1 may play a structural role in signaling, independent of its protein kinase activity. mekk1 knockout mutants display a severe dwarf phenotype, constitutive callose deposition, and constitutive expression of pathogen response genes. This dwarf phenotype was largely rescued by introduction into mekk1 knockout plants of either the MEKK1 (K361M) construct or a nahG transgene that degrades salicylic acid. When treated with pathogenic bacteria, the K361M plants were slightly more susceptible to an avirulent strain of Pseudomonas syringae and showed a delayed hypersensitive response, suggesting a role for MEKK1 kinase activity in this aspect of plant disease resistance. Our results indicate that MEKK1 acts upstream of MPK4 as a negative regulator of pathogen response pathways, a function that may not require MEKK1's full kinase activity.

The protein kinase genes MAP3K epsilon 1 and MAP3K epsilon 2 are required for pollen viability in Arabidopsis thaliana.

We have used reverse-genetic analysis to investigate the function of MAP3K epsilon 1 and MAP3K epsilon 2, a pair of closely related Arabidopsis thaliana genes that encode protein kinases. Plants homozygous for either map3k epsilon 1 or map3k epsilon 2 displayed no apparent mutant phenotype, whereas the double-mutant combination caused pollen lethality. Transmission of the double-mutant combination through the female gametophyte was normal. Tetrad analysis performed using the Arabidopsis quartet mutation demonstrated that the pollen-lethal phenotype segregated at meiosis with the map3k epsilon 1;map3k epsilon 2 genotype. We used transmission electron microscopy to determine that double-mutant pollen grains develop plasma membrane irregularities following pollen mitosis I. Analysis of the subcellular localization of a yellow fluorescent protein (YFP):MAP3Kepsilon1 fusion protein using confocal microscopy and biochemical fractionation indicated that a substantial portion of the MAP3Kepsilon1 present in Arabidopsis cells is localized to the plasma membrane. Taken together, our results suggest that MAP3Kepsilon1 is required for the normal functioning of the plasma membrane in developing Arabidopsis pollen.

Characterization of T-DNA insertion sites in Arabidopsis thaliana and the implications for saturation mutagenesis.

A key component of a sound functional genomics infrastructure is the availability of a knockout mutant for every gene in the genome. A fruitful approach to systematically knockingout genes in the plant Arabidopsis thaliana has been the use of transferred-DNA (T-DNA) from Agrobacterium tumefaciens as an insertional mutagen. One of the assumptions underlying the use of T-DNA as a mutagen is that the insertion of these DNA elements into the Arabidopsis genome occurs at randomly selected locations. We have directly investigated the distribution of T-DNA insertions sites in populations of transformed Arabidopsis using two different approaches. To begin with, we utilized a polymerase chain reaction (PCR) procedure to systematically catalog the precise locations of all the T-DNA elements inserted within a 65 kb segment of chromosome IV. Of the 47 T-DNA insertions identified, 30% were found within the coding regions of genes. We also documented the insertion of T-DNA elements within the centromeric region of chromosome IV. In addition to these targeted T-DNA screens, we also mapped the genomic locations of 583 randomly chosen T-DNA elements by sequencing the genomic DNA flanking the insertion sites from individual T-DNA-transformed lines. 35% of these randomly chosen T-DNA insertions were located within the coding regions of genes. For comparison, coding sequences account for 44% of the Arabidopsis genome. Our results demonstrate that there is a small bias towards recovering T-DNA insertions within intergenic regions. However, this bias does not limit the utility of T-DNA as an effective insertional mutagen for use in reverse-genetic strategies.

An Arabidopsis mitogen-activated protein kinase kinase kinase gene family encodes essential positive regulators of cytokinesis.

The signal transduction pathways that control cytokinesis in plants are largely uncharacterized. Here, we provide genetic evidence that mitogen-activated protein kinase kinase kinases (MAPKKKs) play a role in the control of plant cell division. Using a reverse-genetic approach, we isolated plants carrying knockout alleles of the Arabidopsis MAPKKK genes ANP1, ANP2, and ANP3. The resulting single-mutant plants displayed no obvious abnormal phenotypes; two of the three double-mutant combinations displayed defects in cell division and growth; and the triple-mutant combination was not transmitted through either male or female gametes. The molecular and structural phenotypes displayed by the double mutants support a model in which the ANP family of MAPKKKs positively regulates cell division and growth and may negatively regulate stress responses.