The Activation of the Potato PR-10a Gene Requires the Phosphorylation of the Nuclear Factor PBF-1

The pathogenesis-related gene PR-10a (formerly STH[middot]2) is induced in various organs of potato after wounding, elicitor treatment, or infection by Phytophthora infestans. Deletion analysis of the promoter of the PR-10a gene enabled us to identify a 50-bp region, located between positions -155 and -105, necessary for the elicitor responsiveness of the [beta]-glucuronidase reporter gene in transgenic potato plants. Within this region, a 30-bp sequence, located between positions -135 and -105, was necessary for the activation of the promoter by the elicitor. However, strong promoter activity after elicitor treatment required the presence of a 20-bp sequence located between positions -155 and -135. The region between -135 and -105 was specifically recognized by two nuclear factors, PBF-1 (PR-10a Binding Factor 1) and PBF-2, and binding of PBF-1 was coordinated with the accumulation of the PR-10a mRNA. Gel shift assays using nuclear extracts pretreated with sodium deoxycholate or alkaline phosphatase suggested that PBF-1 is a multimeric factor in which at least one of the constituent proteins can be phosphorylated. Treatment with alkaline phosphatase also indicated that binding of PBF-1 is positively regulated by phosphorylation and that it is phosphorylated only in tissues in which PR-10a is expressed. The use of protein phosphatase and kinase inhibitors in vivo provided additional evidence that wounding and elicitor treatment induce the phosphorylation of PBF-1 and that this phosphorylation is associated with gene activation.     

Role of Jasmonates in the Elicitor- and Wound-Inducible Expression of Defense Genes in Parsley and Transgenic Tobacco

Jasmonates have been proposed to be signaling intermediates in the wound and/or elicitor-activated expression of plant defense genes. We used parsley (Petroselinum crispum) cell cultures and transgenic tobacco (Nicotiana tabacum) plants expressing 4CL1-GUS gene fusions to investigate the potential role played by jasmonates in mediating the wound and/or elicitor activation of phenylpropanoid and other defense-related genes. Jasmonates and [alpha]-linolenic acid strongly induced the expression of 4CL in a dose-dependent manner in parsley cells; methyl jasmonate also activated the coordinate expression of other phenylpropanoid genes and the accumulation of furanocoumarin phytoalexins. However, the response of the cells to optimal methyl jasmonate concentrations was distinct quantitatively and qualitatively from the response of elicitor-treated cells. In transgenic tobacco wound-inducible tobacco 4CL genes and a 4CL1 promoter-GUS transgene were responsive to jasmonates and [alpha]-linolenic acid in a dose-dependent manner. Pre-treatment of parsley cells or tobacco leaves with a lipoxygenase inhibitor reduced their responsiveness to the elicitor and to wounding. These results show that the elicitor response in parsley cells can be partially mimicked by jasmonate treatment, which supports a role for jasmonates in mediating wound-induced expression of 4CL and other phenylpropanoid genes.     

Solution structure of the C4 zinc finger domain of HDM2

HDM2 is a ubiquitin E3 ligase that is a key negative regulator of the tumor suppressor p53. Here, we report the determination of the solution structure of the C4 zinc finger domain of HDM2 using multidimensional NMR. The HDM2 C4 zinc finger domain has a fold consisting of a 3(10) helix followed by four beta-strands, which shares significant structural similarity to the zinc ribbon protein family. Family based sequence analysis identified two putative binding sites, one of which resembles an RNA binding motif.     

A pathogen- and salicylic acid-induced WRKY DNA-binding activity recognizes the elicitor response element of the tobacco class I chitinase gene promoter

Using a simple oligo selection procedure, we have previously identified a tobacco sequence-specific DNA-binding activity, TDBA12, that increases markedly during the tobacco mosaic virus (TMV)-induced hypersensitive response (HR). Based on the binding specificity and the two cDNA clones isolated, TDBA12 is related to a novel class of DNA-binding factors containing WRKY domains. In the present study, we report that TDBA12 could be induced not only by TMV infection but also by treatment with salicylic acid (SA) or its biologically active analogs capable of inducing pathogenesis-related (PR) genes and enhanced resistance. TDBA12 was sensitive to temperature and the protein dissociating agent sodium deoxycholate, suggesting that it may be a multimeric factor in which protein–protein interaction is important for the enhanced DNA-binding activity. Pre-treatment of nuclear extracts with alkaline phosphatase abolished TDBA12, suggesting that protein phosphorylation is important for its high DNA-binding activity. TDBA12 specifically recognized the elicitor response element of the tobacco class I basic chitinase gene promoter. The increase in the levels of TDBA12 following TMV infection or SA treatment preceded the induced expression of the tobacco chitinase gene. These results strongly suggest that certain WRKY DNA-binding proteins may be activated by enhanced protein phosphorylation and regulate inducible expression of defense-related genes during pathogen- and SA-induced plant defense responses.     

Positive and negative cooperativity of modularly assembled zinc fingers

One simple and widespread method to create engineered zinc fingers targeting the desired DNA sequences is to modularly assemble multiple finger modules pre-selected to recognize each DNA triplet. However, it has become known that a sufficient DNA binding affinity is not always obtained. In order to create successful zinc finger proteins, it is important to understand the context-dependent contribution of each finger module to the DNA binding ability of the assembled zinc finger proteins. Here, we have created finger-deletion mutants of zinc finger proteins and examined the DNA bindings of these zinc fingers to clarify the contributions of each finger module. Our results indicate that not only a positive cooperativity but also a context-dependent reduction in the DNA binding activity can be induced by assembling zinc finger modules.     

Zinc fingers in sex determination: only one of the two C. elegans Tra-1 proteins binds DNA in vitro.

The tra-1 gene of Caenorhabditis elegans is a major developmental regulator that promotes female development. Two mRNAs are expressed from the tra-1 locus as a result of alternative mRNA processing. One mRNA encodes a protein with five zinc fingers and the other a protein with only the first two zinc fingers. We have derived a preferred in vitro DNA binding site for the five finger protein by selection from random oligonucleotides. The two finger protein does not bind to DNA in vitro. Moreover, removal of the first two fingers from the five finger protein does not eliminate binding and has little effect on its preferred binding site. We find that a protein sequence amino-terminal to the finger domain also appears to play a role in DNA binding.     

Expression of a RING-HC protein from rice improves resistance to Pseudomonas syringae pv. tomato DC3000 in transgenic Arabidopsis thaliana

A cDNA clone (OsRHC1) was obtained, which encodes a novel RING zinc finger protein sharing similar structural features (multiple transmembrane domains at the N-half; a unique RING zinc finger consensus Cys-X(2)-Cys-X(11)-Cys-X-His-X(3)-Cys-X(2)-Cys-X(6)-Cys-X(2)-Cys at the C terminus) to a group of closely related annotated proteins from both monocots and dicots. OsRHC1 was found to be localized on plasma membrane of rice cells and induced by wounding in rice lines containing Xa loci. Ecotopic expression of the OsRHC1 cDNA from rice (a monocot) in transgenic Arabidopsis thaliana (a dicot) enhanced the defence response toward Pseudomonas syringae pv. tomato DC3000, suggesting that OsRHC1 may confer broad-spectrum disease resistance. The protective effects were neutralized in the presence of MG132 or in an npr1-3 mutation background, indicating that the function of OsRHC1 is dependent on the ubiquitin-mediated protein degradation via the 26S proteasome and the presence of the key defence response regulator NPR1.