Noncanonical mono(ADP-ribosyl)ation of zinc finger SZF proteins counteracts ubiquitination for protein homeostasis in plant immunity.

Protein ADP-ribosylation is a reversible post-translational modification that transfers ADP-ribose from NAD+ onto acceptor proteins. Poly(ADP-ribosyl)ation (PARylation), catalyzed by poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolases (PARGs), which remove the modification, regulates diverse cellular processes. However, the chemistry and physiological functions of mono(ADP-ribosyl)ation (MARylation) remain elusive. Here, we report that Arabidopsis zinc finger proteins SZF1 and SZF2, key regulators of immune gene expression, are MARylated by the noncanonical ADP-ribosyltransferase SRO2. Immune elicitation promotes MARylation of SZF1/SZF2 via dissociation from PARG1, which has an unconventional activity in hydrolyzing both poly(ADP-ribose) and mono(ADP-ribose) from acceptor proteins. MARylation antagonizes polyubiquitination of SZF1 mediated by the SH3 domain-containing proteins SH3P1/SH3P2, thereby stabilizing SZF1 proteins. Our study uncovers a noncanonical ADP-ribosyltransferase mediating MARylation of immune regulators and underpins the molecular mechanism of maintaining protein homeostasis by the counter-regulation of ADP-ribosylation and polyubiquitination to ensure proper immune responses.

Genome-wide analysis and stress-responsive expression of CCCH zinc finger family genes in Brassica rapa.

BACKGROUND: Ubiquitous CCCH nucleic acid-binding motif is found in a wide-variety of organisms. CCCH genes are involved in plant developmental processes and biotic and abiotic stress responses. Brassica rapa is a vital economic crop and classical model plant of polyploidy evolution, but the functions of CCCH genes in B. rapa are unclear. RESULTS: In this study, 103 CCCH genes in B. rapa were identified. A comparative analysis of the chromosomal position, gene structure, domain organization and duplication event between B. rapa and Arabidopsis thaliana were performed. Results showed that CCCH genes could be divided into 18 subfamilies, and segmental duplication might mainly contribute to this family expansion. C-X7/8-C-X5-C3-H was the most commonly found motif, but some novel CCCH motifs were also found, along with some loses of typical CCCH motifs widespread in other plant species. The multifarious gene structures and domain organizations implicated functional diversity of CCCH genes in B. rapa. Evidence also suggested functional redundancy in at least one subfamily due to high conservation between members. Finally, the expression profiles of subfamily-IX genes indicated that they are likely involved in various stress responses. CONCLUSION: This study provides the first genome-wide characterization of the CCCH genes in B. rapa. The results suggest that B. rapa CCCH genes are likely functionally divergent, but mostly involved in plant development and stress response. These results are expected to facilitate future functional characterization of this potential RNA-binding protein family in Brassica crops.

OsWRKY80-OsWRKY4 Module as a Positive Regulatory Circuit in Rice Resistance Against Rhizoctonia solani.

BACKGROUND: Plant WRKY transcription factors play pivotal roles in diverse biological processes but most notably in plant defense response to pathogens. Sheath blight represents one of the predominant diseases in rice. However, our knowledge about the functions of WRKY proteins in rice defense against sheath blight is rather limited. RESULTS: Here we demonstrate that the expression of Oryza sativa WRKY80 gene (OsWRKY80) is rapidly and strongly induced upon infection of Rhizoctonia solani, the causal agent of rice sheath blight disease. OsWRKY80 expression is also induced by exogenous jasmonic acid (JA) and ethylene (ET), but not by salicylic acid (SA). OsWRKY80-GFP is localized in the nuclei of onion epidermal cells in a transient expression assay. Consistently, OsWRKY80 exhibits transcriptional activation activity in a GAL4 assay in yeast cells. Overexpression of OsWRKY80 in rice plants significantly enhanced disease resistance to R. solani, concomitant with elevated expression of OsWRKY4, another positive regulator in rice defense against R. solani. Suppression of OsWRKY80 by RNA interference (RNAi), on the other hand, compromised disease resistance to R. solani. Results of yeast one-hybrid assay and transient expression assay in tobacco cells have revealed that OsWRKY80 specifically binds to the promoter regions of OsWRKY4, which contain W-box (TTGAC[C/T]) or W-box like (TGAC[C/T]) cis-elements. CONCLUSIONS: We propose that OsWRKY80 functions upstream of OsWRKY4 as an important positive regulatory circuit that is implicated in rice defense response to sheath blight pathogen R. solani.

Molecular and cellular characterization of GA-Stimulated Transcripts GASA4 and GASA6 in Arabidopsis thaliana.

GA and ABA play antagonistic roles in numerous cellular processes essential for growth, development, and stress responses. GASA4 and GASA6 belong to a family of GA-Stimulated transcripts in Arabidopsis, known as GA-inducible and ABA-repressible. We have found that GASA4 and GASA6 expression is likely mediated through a repressor of GA responses, GA INSENSITIVE (GAI) protein. Moreover, GASA4 and GASA6 are in general up regulated by growth hormones (auxin, BR, cytokinin, and GA) and down regulated by stress hormones (ABA, JA, and SA), indicating a role of GASA4 and GASA6 in hormone crosstalk. Genetic analyses show that suppression of both GASA4 and GASA6 causes late flowering, while over-expression of GASA6 causes early flowering in Arabidopsis. GASA family members encode small polypeptides sharing common structural features: an N-terminal signal peptide, a highly divergent intermediate region, and a conserved C-terminal domain containing 12 conserved cysteines. Despite the presence of a signal peptide, it has not been determined whether or not GASA4 and GASA6 can be processed in vivo. By using imaging and immunological analyses, we show that the N-terminal signal peptide is cleaved as predicted, and the cleavage is important for proper sub-cellular localization of GASA4 and GASA6.

Plant Tandem CCCH Zinc Finger Proteins Interact with ABA, Drought, and Stress Response Regulators in Processing-Bodies and Stress Granules.

Although multiple lines of evidence have indicated that Arabidopsis thaliana Tandem CCCH Zinc Finger proteins, AtTZF4, 5 and 6 are involved in ABA, GA and phytochrome mediated seed germination responses, the interacting proteins involved in these processes are unknown. Using yeast two-hybrid screens, we have identified 35 putative AtTZF5 interacting protein partners. Among them, Mediator of ABA-Regulated Dormancy 1 (MARD1) is highly expressed in seeds and involved in ABA signal transduction, while Responsive to Dehydration 21A (RD21A) is a well-documented stress responsive protein. Co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays were used to confirm that AtTZF5 can interact with MARD1 and RD21A in plant cells, and the interaction is mediated through TZF motif. In addition, AtTZF4 and 6 could also interact with MARD1 and RD21A in Y-2-H and BiFC assay, respectively. The protein-protein interactions apparently take place in processing bodies (PBs) and stress granules (SGs), because AtTZF5, MARD1 and RD21A could interact and co-localize with each other and they all can co-localize with the same PB and SG markers in plant cells.

Tandem CCCH zinc finger proteins in plant growth, development and stress response.

Cysteine3Histidine (CCCH)-type zinc finger proteins comprise a large family that is well conserved across eukaryotes. Among them, tandem CCCH zinc finger proteins (TZFs) play critical roles in mRNA metabolism in animals and yeast. While there are only three TZF members in humans, a much higher number of TZFs has been found in many plant species. Notably, plant TZFs are over-represented by a class of proteins containing a unique TZF domain preceded by an arginine (R)-rich (RR) motif, hereafter called RR-TZF. Recently, there have been a large number of reports indicating that RR-TZF proteins can localize to processing bodies (P-bodies) and stress granules (SG), two novel cytoplasmic aggregations of messenger ribonucleoprotein complexes (mRNPs), and play critical roles in plant growth, development and stress response, probably via RNA regulation. This review focuses on the classification and most recent development of molecular, cellular and genetic analyses of plant RR-TZF proteins.

Phytochromes A1 and B1 have distinct functions in the photoperiodic control of flowering in the obligate long-day plant Nicotiana sylvestris.

The obligate long-day plant Nicotiana sylvestris with a nominal critical day length of 12 h was used to dissect the roles of two major phytochromes (phyA1 and phyB1) in the photoperiodic control of flowering using transgenic plants under-expressing PHYA1 (SUA2), over-expressing PHYB1 (SOB36), or cosuppressing the PHYB1 gene (SCB35). When tungsten filament lamps were used to extend an 8 h main photoperiod, SCB35 and SOB36 flowered earlier and later, respectively, than wild-type plants, while flowering was greatly delayed in SUA2. These results are consistent with those obtained with other long-day plants in that phyB has a negative role in the control of flowering, while phyA is required for sensing day-length extensions. However, evidence was obtained for a positive role for PHYB1 in the control of flowering. Firstly, transgenic plants under-expressing both PHYA1 and PHYB1 exhibited extreme insensitivity to day-length extensions. Secondly, flowering in SCB35 was completely repressed under 8 h extensions with far-red-deficient light from fluorescent lamps. This indicates that the dual requirement for both far-red and red for maximum floral induction is mediated by an interaction between phyA1 and phyB1. In addition, a diurnal periodicity to the sensitivity of both negative and positive light signals was observed. This is consistent with existing models in which photoperiodic time measurement is not based on the actual measurement of the duration of either the light or dark period, but rather the coincidence of endogenous rhythms of sensitivity - both positive and negative - and the presence of light cues.