Screening and Identification of Candidate GUN1-Interacting Proteins in Arabidopsis thaliana

Chloroplasts are semi-autonomous organelles governed by the precise coordination between the genomes of their own and the nucleus for functioning correctly in response to developmental and environmental cues. Under stressed conditions, various plastid-to-nucleus retrograde signals are generated to regulate the expression of a large number of nuclear genes for acclimation. Among these retrograde signaling pathways, the chloroplast protein GENOMES UNCOUPLED 1 (GUN1) is the first component identified. However, in addition to integrating aberrant physiological signals when chloroplasts are challenged by stresses such as photooxidative damage or the inhibition of plastid gene expression, GUN1 was also found to regulate other developmental processes such as flowering. Several partner proteins have been found to interact with GUN1 and facilitate its different regulatory functions. In this study, we report 15 possible interacting proteins identified through yeast two-hybrid (Y2H) screening, among which 11 showed positive interactions by pair-wise Y2H assay. Through the bimolecular fluorescence complementation assay in Arabidopsis protoplasts, two candidate proteins with chloroplast localization, DJC31 and HCF145, were confirmed to interact with GUN1 in planta. Genes for these GUN1-interacting proteins showed different fluctuations in the WT and gun1 mutant under norflurazon and lincomycin treatments. Our results provide novel clues for a better understanding of molecular mechanisms underlying GUN1-mediated regulations.

Description of AtCAX4 in Response to Abiotic Stress in Arabidopsis

High-capacity tonoplast cation/H+ antiport in plants is partially mediated by a family of CAX transporters. Previous studies have reported that CAX activity is affected by an N-terminal autoinhibitory region. CAXs may be present as heterodimers in plant cells, and this phenomenon necessitates further study. In this study, we demonstrate that there is an interaction between CAX4 and CAX1 as determined by the use of a yeast two-hybrid system and a bimolecular fluorescence complementation assay. More specifically, the N-terminal of CAX4 interacts with CAX1. We further observed the over-expression and either a single or double mutant of CAX1 and CAX4 in response to abiotic stress in Arabidopsis. These results suggest that CAX1 and CAX4 can interact to form a heterodimer, and the N-terminal regions of CAX4 play important roles in vivo; this may provide a foundation for a deep study of CAX4 function in the future.

Maize AKINβγ Proteins Interact with P8 of Rice Black Streaked Dwarf Virus and Inhibit Viral Infection

Rice black streaked dwarf virus (RBSDV) is an important agent causing maize rough dwarf disease, whereas the host factors responding to RBSDV infection are poorly understood. To uncover the molecular interactions between RBSDV and maize, a yeast two-hybrid screen of a maize cDNA library was carried out using the viral P8 protein as a bait. ZmAKINβγ-1 and ZmAKINβγ-2 (βγ subunit of Arabidopsis SNF1 kinase homolog in maize) possessing high sequence similarities (encoded by two gene copies) were identified as interaction partners. Their interactions with P8 were confirmed in both Nicotiana benthamiana cells and maize protoplasts by bimolecular fluorescence complementation assay. The accumulation levels of ZmAKINβγ mRNAs were upregulated at the stage of the viral symptoms beginning to appear and then downregulated. ZmAKINβγs are putative regulatory subunits of the SnRK1 complex, a core regulator for energy homeostasis. Knockdown of ZmAKINβγs in maize regulated the expression levels of the genes involved in sugar synthesis or degradation, and also the contents of both glucose and sucrose. Importantly, downregulation of ZmAKINβγs expressions facilitated the accumulation of RBSDV in maize. These results implicate a role of ZmAKINβγs in the regulation of primary carbohydrate metabolism, and in the defense against RBSDV infection.

Cytosolic TaGAPC2 Enhances Tolerance to Drought Stress in Transgenic Arabidopsis Plants

Drought is a major natural disaster that seriously affects agricultural production, especially for winter wheat in boreal China. As functional proteins, the functions and mechanisms of glyceraldehyde-3-phosphate dehydrogenase in cytoplasm (GAPCs) have remained little investigated in wheat subjected to adverse environmental conditions. In this study, we cloned and characterized a GAPC isoform TaGAPC2 in wheat. Over-expression of TaGApC2-6D in Arabidopsis led to enhanced root length, reduced reactive oxygen species (ROS) production, and elevated drought tolerance. In addition, the dual-luciferase assays showed that TaWRKY28/33/40/47 could positively regulate the expression of TaGApC2-6A and TaGApC2-6D. Further results of the yeast two-hybrid system and bimolecular fluorescence complementation assay (BiFC) demonstrate that TaPLDδ, an enzyme producing phosphatidic acid (PA), could interact with TaGAPC2-6D in plants. These results demonstrate that TaGAPC2 regulated by TaWRKY28/33/40/47 plays a crucial role in drought tolerance, which may influence the drought stress conditions via interaction with TaPLDδ. In conclusion, our results establish a new positive regulation mechanism of TaGAPC2 that helps wheat fine-tune its drought response.

Identification of client iron–sulfur proteins of the chloroplastic NFU2 transfer protein in Arabidopsis thaliana

Iron–sulfur (Fe-S) proteins have critical functions in plastids, notably participating in photosynthetic electron transfer, sulfur and nitrogen assimilation, chlorophyll metabolism, and vitamin or amino acid biosynthesis. Their maturation relies on the so-called SUF (sulfur mobilization) assembly machinery. Fe-S clusters are synthesized de novo on a scaffold protein complex and then delivered to client proteins via several transfer proteins. However, the maturation pathways of most client proteins and their specificities for transfer proteins are mostly unknown. In order to decipher the proteins interacting with the Fe-S cluster transfer protein NFU2, one of the three plastidial representatives found in Arabidopsis thaliana, we performed a quantitative proteomic analysis of shoots, roots, and seedlings of nfu2 plants, combined with NFU2 co-immunoprecipitation and binary yeast two-hybrid experiments. We identified 14 new targets, among which nine were validated in planta using a binary bimolecular fluorescence complementation assay. These analyses also revealed a possible role for NFU2 in the plant response to desiccation. Altogether, this study better delineates the maturation pathways of many chloroplast Fe-S proteins, considerably extending the number of NFU2 clients. It also helps to clarify the respective roles of the three NFU paralogs NFU1, NFU2, and NFU3.

The WRKY53 transcription factor enhances stilbene synthesis and disease resistance by interacting with MYB14 and MYB15 in Chinese wild grape

Resveratrol is notable not only for its functions in disease resistance in plants but also for its health benefits when it forms part of the human diet. Identification of new transcription factors helps to reveal the regulatory mechanisms of stilbene synthesis. Here, the WRKY53 transcription factor was isolated from the Chinese wild grape, Vitis quinquangularis. Vqwrky53 was expressed in a variety of tissues and responded to powdery mildew infection and to exogenous hormone application. VqWRKY53 was located in the nucleus and had transcriptional activation activity in yeast. A yeast two-hybrid assay and a bimolecular fluorescence complementation assay confirmed that VqWRKY53 interacted physically with VqMYB14 and VqMYB15, which have previously been reported to regulate stilbene synthesis. When Vqwrky53 was overexpressed in grape leaves, the expression of VqSTS32 and VqSTS41 and the content of stilbenes were increased. A yeast one-hybrid assay demonstrated that VqWRKY53 could bind directly to the promoters of STS genes. Overexpression of Vqwrky53 activated β-glucuronidase expression, driven by STS promoters, and co-expressing Vqwrky53 with VqMYB14 and VqMYB15 showed stronger regulatory functions. Heterologous overexpression of Vqwrky53 in Arabidopsis accelerated leaf senescence and disease resistance to PstDC3000.

Soybean Cytochrome b5 Is a Restriction Factor for Soybean Mosaic Virus

Soybean mosaic virus (SMV) is one of the most destructive viral diseases in soybeans (Glycine max). In this study, an interaction between the SMV P3 protein and cytochrome b5 was detected by yeast two-hybrid assay, and bimolecular fluorescence complementation assay showed that the interaction took place at the cell periphery. Further, the interaction was confirmed by co-immunoprecipitation analysis. Quantitative real-time polymerase chain reaction analysis revealed that GmCYB5 gene was differentially expressed in resistant and susceptible soybean plants after inoculation with SMV-SC15 strain. To test the involvement of this gene in SMV resistance, the GmCYB5 was silenced using a bean pod mottle virus (BPMV)-based vector construct. Results showed that GmCYB5-1 was 83% and 99% downregulated in susceptible (NN1138-2) and resistant (RN-9) cultivars, respectively, compared to the empty vector-treated plants. Silencing of GmCYB5 gene promotes SMV replication in soybean plants. Our results suggest that during SMV infection, the host CYB5 protein targets P3 protein to inhibit its proliferation. Taken together, these results suggest that CYB5 is an important factor in SMV infection and replication in soybeans, which could help soybean breeders develop SMV resistant soybean cultivars.