Plant immune responses are triggered by pattern recognition receptors that detect conserved pathogen-associated molecular patterns (PAMPs) or by resistance (R) proteins recognizing isolate-specific pathogen effectors. We show that in barley, intracellular mildew A (MLA) R proteins function in the nucleus to confer resistance against the powdery mildew fungus. Recognition of the fungal avirulence A10 effector by MLA10 induces nuclear associations between receptor and WRKY transcription factors. The identified WRKY proteins act as repressors of PAMP-triggered basal defense. MLA appears to interfere with the WRKY repressor function, thereby de-repressing PAMP-triggered basal defense. Our findings reveal a mechanism by which these polymorphic immune receptors integrate distinct pathogen signals.

WRKY proteins comprise a large family of transcription factors. Despite their dramatic diversification in plants, WRKY genes seem to have originated in early eukaryotes. The cognate DNA-binding site of WRKY factors is well defined, but determining the roles of individual family members in regulating specific transcriptional programs during development or in response to environmental signals remains daunting. This review summarises the recent advances made in starting to unravel the various functions controlled by WRKY proteins.Addresses

Regulatory proteins play critical roles in controlling the kinetics of various cellular processes during the entire life span of an organism. Leaf senescence, an integral part of the plant developmental program, is Wne-tuned by a complex transcriptional regulatory network ensuring a successful switch to the terminal life phase. To expand our understanding on how transcriptional control coordinates leaf senescence, we characterized AtWRKY70, a gene encoding a WRKY transcription factor that functions as a negative regulator of developmental senescence. To gain insight into the interplay of senescence and plant defense signaling pathways, we employed a collection of mutants, allowing us to speciWcally deWne the role of AtWRKY70 in the salicylic acid-mediated signaling cascades and to further dissect the cross-talk of signal transduction pathways during the onset of senescence in Arabidopsis thaliana. Our results provide strong evidence that AtWRKY70 inXuences plant senescence and defense signaling pathways. These studies could form the basis for further unraveling of these two complex interlinked regulatory networks.

SummaryThe sfr6-1 mutant of Arabidopsis thaliana was identified previously on the basis of its failure to undergo acclimation to freezing temperatures following exposure to low positive temperatures. This failure is attributed to a defect in the pathway leading to cold on-regulated (COR) gene expression via CBF (C-box binding factor) transcription factors. We identified a region of chromosome 4 containing SFR6 by positional mapping. Fine mapping of the sfr6-1 mutation proved impossible as the locus resides very close to the centromere. Therefore, we screened 380 T-DNA lines with insertions in genes within the large region to which sfr6-1 mapped. This resulted in the identification of two further mutant alleles of SFR6 (sfr6-2 and sfr6-3); like the original sfr6-1 mutation, these disrupt freezing tolerance and COR gene expression. To determine the protein sequence, we cloned an SFR6 cDNA based on the predicted coding sequence, but this offered no indication as to the mechanism by which SFR6 acts. The SFR6 gene itself is not strongly regulated by cold, thus discounting regulation of SFR6 activity at the transcriptional level. We show that over-expression of CBF1 or CBF2 transcription factors, which constitutively activate COR genes in the wild-type, cannot do so in sfr6-1. We demonstrate that CBF protein accumulates to wild-type levels in response to cold in sfr6-1. These results indicate a role for the SFR6 protein in the CBF pathway -downstream of CBF translation. The fact that the SFR6 protein is targeted to the nucleus may suggest a direct role in modulating gene expression.

Designer transcription-activator like effectors (TALEs) is a promising technology and made it possible to edit genomes with higher specificity. Such specific engineering and gene regulation technologies are also being developed using RNA-binding proteins like PUFs and PPRs. The main feature of TALEs, PUFs and PPRs is their repetitive DNA/RNA-binding domains which have single nucleotide binding specificity. Available kits today allow researchers to assemble these repetitive domains in any combination they desire when generating TALEs for gene targeting and editing. However, PCR amplifications of such repetitive DNAs are highly problematic as these mostly fail, generating undesired artifact products or deletions. Here we describe the molecular mechanisms leading to these artifacts. We tested our models also in plasmid templates containing one copy versus two copies of GFP-coding sequence arranged as either direct or inverted repeats. Some limited solutions in amplifying repetitive DNA regions are described.

The simplified method works as efficiently as the previously reported protocols and significantly reduces the workload, cost and time. Additionally, the protocol reduces the risk of large scale contaminations involving GMOs. Most importantly, many more independent transformations per day can be performed using this modified protocol.

SummaryIn the course of several different projects, we came to realize that there is a significant amount of untapped potential in the publicly available T-DNA insertion lines. In addition to the GABI-Kat lines, which were designed specifically for activation tagging, lines from the SAIL and FLAGdb collections are also useful for this purpose. As well as the 35S promoter chosen for activation tagging in GABI-Kat lines, we found that the 1¢2¢ bidirectional promoter is capable of activating expression of flanking genomic sequences in both GABI-Kat and SAIL lines. Thus these lines have added potential for activation tagging. We also show that these lines are capable of generating antisense transcripts and so have the potential to be used for suppression (loss/reduction of function) studies. By virtue of weak terminator sequences in some T-DNA constructs, transcript read-through from selectable markers is also possible, which again has the potential to be exploited in activation/ suppression studies. Finally, we show that, by selecting and characterizing lines in which the T-DNA insertions are present specifically within introns of a target gene, an allelic series of mutants with varying levels of reduced expression can be generated, due to differences in efficiency of intron splicing. Taken together, our analyses demonstrate that there is a wealth of untapped potential within existing insertion lines for studies on gene function, and the effective exploitation of these resources is discussed.

SummaryThe RB7 matrix attachment region (MAR), when flanking a uidA (GUS) reporter gene, has been previously shown to increase uidA gene expression by 60-fold in stably transformed tobacco suspension cell lines. We have now used the same co-transformation procedure to determine the effect of flanking MARs on uidA gene expression in tobacco plants. The neomycin phosphotransferase selection gene and uidA reporter gene on separate plasmids were co-transformed into seedlings by microprojectile bombardment. In primary transgenic plants, the average uidA expression in plants with MARs was twofold greater than in control plants without MARs, but there was no effect on variation of expression. GUS activity was not proportional to the number of integrated uidA transgenes over the entire range of copy numbers. However, in the lower part of the copy number range, MAR lines show a tendency for expression to increase with copy number. Transgene expression in backcross progenies of the MARcontaining lines averaged threefold higher than in control progenies. MARs also reduced the loss of transgene expression in the BC 1 generation. Sixty-three per cent of the 21 MAR-containing primary transformants, but only 20% of the 14 control primary transformants, produced backcross progenies in which no loss of transgene expression was observed. These observations are discussed in the context of homology-dependent gene silencing.