Phylogenetic analysis of barley WRKY proteins and characterization of HvWRKY1 and -2 as repressors of the pathogen-inducible gene HvGER4c.

The WRKY transcription factors belong to an evolutionarily conserved superprotein family predominantly present in the plant kingdom. WRKY proteins of barley are not yet fully annotated and most of them are not functionally characterized. We performed a genome-wide identification of WRKY members based on the recently accessible barley draft genome sequence and full-length cDNA datasets. As a result, 34 novel putative proteins have been identified which extend the existing list for barley WRKYs to 94. Phylogenetic analysis of the WRKY domains allowed ranking into three groups (I, II, III), with an expansion in group III in monocots. Two members of subgroup IIa, the wound and pathogen-inducible HvWRKY1 and HvWRKY2, are known as negative defense regulators. Here, we demonstrate that both transcription factors repress the activity of the powdery mildew-induced promoter of HvGER4c, a germin-like defense-related protein. The repression did not require the negative defense regulator MLO nor was it affected by the presence of the R protein MLA12. Moreover, the expression of the Arabidopsis ortholog AtWRKY40 in barley compromised basal resistance to powdery mildew, providing evidence for functional conservation of sequence-related WRKY proteins across monocots and dicots.

Molecular characterization of Na+/H+ antiporters (ZmNHX) of maize (Zea mays L.) and their expression under salt stress.

Six full-length gene transcripts ZmNHX1-6 from Zea mays L. that were homologous to tonoplast-associated Na+/H+ antiporter were identified. The deduced transcripts code 538-545 amino acids and share a high homology with those of putative tonoplast-associated Na+/H+ antiporters of higher plants, ranging from 78% homology with that of Arabidopsis thaliana (AtNHX1) to 63% with that of tomato (LeNHX1). On the other hand, the homology of the identified transcripts with those of plasma membrane or mitochondrial membrane-associated Na+/H+ antiporters was low. An amiloride-binding site in transmembrane domain M4 was predicted. The phylogenetic analysis grouped the six isoforms into two groups. ZmNHX1,2,6 form one group together with Arabidopsis AtNHX1,2, rice OsNHX and wheat TaNHX2. The second group, ZmNHX3-5, clusters with Arabidopsis AtNHX4-6 and tomato LeNHX2. The expression of ZmNHX isoforms at the mRNA Level showed an organ and salt-specific pattern. In addition, a genotype-specific expression pattern of the ZmNHX isoforms was detected. One genotype was a maize inbred line with high Na+ exclusion at the root surface and the level of xylem parenchyma. For the root tissue of this inbred line, a linear response of ZmNHX to NaCl concentrations in root medium ranging from 1 to 100 mM was detected using real-time PCR. Conversely, there was no salt response of ZmNHX for the shoot of the same plant. No salt response of ZmNHX was detected for maize F1 hybrid Pioneer 3906 with moderate Na+ exclusion. The relationship of the expression of ZmNHX with salt resistance of maize is discussed.

Identification of powdery mildew-induced barley genes by cDNA-AFLP: functional assessment of an early expressed MAP kinase.

Gene expression analysis by cDNA-AFLP in barley ( Hordeum vulgare L.) after powdery mildew ( Blumeria graminis f.sp. hordei , Bgh ) inoculation revealed 615 (3.7%) of 16 500 screened cDNA fragments being differentially regulated 4 and/or 12 h after inoculation. Of these transcript derived fragments (TDFs), 120 were sequenced, and for 28 out of 29 tested, induction was confirmed via RT-PCR. Most TDFs did not show any homology to sequences with known functions, others showed homology to genes involved in primary and secondary metabolism, pathogen response, redox regulation, and signal transduction. TDFs with homology to a MAP kinase ( PWMK1 ), a WRKY transcription factor, a heparanase, an immunophilin, a cytochrome P450, and a receptor-like protein kinase were isolated as full length cDNAs. Knockdown by RNA interference via biolistic delivery of sequence specific double stranded RNA to leaf segments tagged two of these genes as possible candidates being causally involved in the outcome of the barley- Bgh interaction. Knockdown of the receptor-like protein kinase and the WRKY transcription factor increased resistance to the fungus, while knockdown of PWMK1 only led to a slightly enhanced susceptibility of epidermal cells to Bgh . This suggests that the receptor-like protein kinase and the WRKY protein are candidates for negative regulators of powdery mildew resistance. Based on expression analyses, PWMK1 appears to be more generally involved in stress response.