State Key Laboratory of Protein and Plant Gene Research State Key Laboratory of Protein and Plant Gene Research

The State Key Laboratory of Protein and Plant Gene Research (formerly The National Laboratory of Protein Engineering and Plant Genetic Engneering) was first established in January, 1987 and officially recognized by Ministry of Science and Technology in September, 1990. Professor Zhihong Xu is currently serving as the Chairman of the Academic Committee of the Laboratory, and Professor Yuxian Zhu is the Director. The Laboratory has 34 professors, 1 professor-level senior engineer and 6 associate or assistant professors. The lab has three members of the Chinese Academy of Sciences (Professors Zhihong Xu, Yuxian Zhu, Jindong Zhao) and one USAS academy fellow/ 1000-Talent Scientist (Professor Xing-Wang Deng), eight Cheung-kung Scholars, ten NSFC Excellent Young Scientists, five Young 1000-Talent Scientist and one Young Excellent Talent. We have five principal scientists of National Key Basic Research Program over the past a few years. Scientists in the lab has made significant progresses in several important aspects of basic research that resulted in publications in top academic journals such as Nature, Cell, Science, Nature Genetics, Nature Biotechnology and Nature Methods. Altogether, scientists from this Laboratory has published 291 peer-reviewed research articles and review papers, including three in Cell, one each in Nature, Science, Nat Biotechnol., Nat Methods, Nat. Cell Biol. and Nat. Neurosci., two each in Nature Genetics, Nat. Struct. Mol. Biol. and Nat. Chem Biol. We have also published in Genome Res. (2), Mol Cell (1)、Cell Res. (13), Autophagy (1), Nat Commun. (3), Angew. Chem.-Int. Edit. (1), Genome Biol. (1), Proc. Natl. Acad. Sci. U. S. A. (10), Brief Bioinform. (1), Curr Biol. (2), Nucleic Acids Res. (11), Mol Biol Evol (2), Plant Cell (16) and Elife (1). The total SCI impact factors of these published papers are greater than 2100, with an average impact factor 6.79 for each paper. We have published 83 papers in Journals with SCI impact factor greater than 9, that accounted for a more than four-fold increase when compared with the last evaluation period between year 2006 and year 2010.

For example, we found a novel innate immune signaling pathway that is dependent on STAT6. Viruses or cytoplasmic nucleic acids trigger STING (also named MITA/ERIS) to recruit STAT6, leading to STAT6 phosphorylation by TBK1. Phosphorylated STAT6 then dimerizes and translocates to nucleus to induce specific target genes responsible for immune cell homing. Thus, STING functions as an adaptor for the interactions involving MAVS, STAT6 and TBK1, integrating signals from various stimuli. The physiological significance of STAT6 is highlighted by the fact that Stat6–/– mice are highly susceptible to virus infection (CELL, 2011, 147: 436-446). Thus, our study identifies a previously unknown STAT6 activating cascade which plays a critical role in innate immunity against microbial infection.

Through biochemical and genetic screening, we obtained a few dozens of potential checkpoint targets. Among these, Dna2 appears to be a major checkpoint target that plays an important role in stabilizing stalled replication forks. In the Dna2-defective cells, reversed replication forks significantly accumulate. Reversed forks are a kind of pathological structure and they cause fork collapse, which further results in DNA breaks, incomplete DNA synthesis and genomic rearrangement. We find that the S phase checkpoint ATR-Chk2 directly phosphorylates Dna2 at S220 position and thus stabilizes the association of Dna2 and stalled replication forks. Dna2 is flap endonuclease and it cleaves an un-annealed nascent strand end to prevent stalled forks from reversing (CELL, 2012, 149: 1221-1232). This finding solves a long-standing question how the S-phase checkpoint prevents stalled forks from reversing, and it is a breakthrough in this research field. This finding also suggests that replisome should be a critical target in stabilizing stalled replication forks.

We revealed the mechanism that plant endogenous genes especially the protein coding genes averted the post-transcriptional gene silencing machine, a vital cell immune system in plant. We firstly performed a cosuppression-based suppressor screen in ArabidopsisEIN3ox background and obtained three genes essential for either 3’ or 5’ cytoplasmic mRNA decay (SKI2/SKI3, EIN5), which indicated that cytoplasmic RNA decay pathways suppressed transgene silencing. Those two RNA decay pathways are highly conserved in eukaryotes, and the Arabidopsis ski2 or ein5 mutant is normal in development, suggesting they are functional redundant in plant growth development (SCIENCE, 2015, 348: 120-123). However, plants are embryo lethal in the absence of both EIN5 and SKI2. The partially loss-of-function ein5 ski2 mutant displays severe growth defects, including delay development, decreased activity in shoot apical meristem. Accordingly, dramatic transcriptomic alteration was also observed in ein5 ski2 mutant. Interestingly, almost all the growth defects and transcriptomic changes were substantially suppressed by the RNAi pathway mutants. By using small RNA sequencing, 441 coding-transcripts-derived siRNAs (ct-siRNAs) were highly accumulated upon the dysfunction of EIN5 and SKI2. Those ct-siRNAs are 21-22 nt in length, whose production are dependent on RDR6-DCL2/4 pathway and functionally dependent on AGO1. Further analysis suggested that at least part of the ct-siRNAs interfered with their cognate genes expression, resulting in the multiple growth defects.

We reported a novel branch of ethylene signaling pathway (Cell, 2015, 163: 670-683). EIN2, whose null mutant was completely insensitive to ethylene, was regarded as an essential central positive regulator. Years ago, our work reveal that ER-located EIN2 translocate to nucleus to active ethylene response through a “cleave and shuttle” model. Recently, wefind that EIN2 imposes the translational repression of EBF1 and EBF2 mRNA. Our study show that the EBF1/2 3’ untranslated regions (3’UTRs) mediate EIN2-directed translational repression, and identify multiple poly-uridylates (PolyU) motifs as functional cis-elements of 3’UTRs. Furthermore, we demonstrate that ethylene induces EIN2 to associate with 3’UTRs and target EBF1/2 mRNA to cytoplasmic processing-body (P-body) through interacting with multiple P-body factors, including EIN5 and PABs. Our study illustrates translational regulation as a key step in ethylene signaling, and presents mRNA 3’UTR functioning as a “signal transducer” to sense and relay cellular signaling in plants.

One of the most important challenges in the research of life sciences is to attribute physiological functions precisely to responsible genes. Taking advantage of the targeted genome editing technologies, we developed a focused CRISPR/Cas9-based lentiviral library in human cells and a method of gene identification based on functional screening and high-throughput sequencing analysis (Nature, 2014, 509:487-491). Using knockout library screens, we successfully identified the host components essential for the intoxication of cells by two bacterial toxins, which were confirmed by functional validation. The broad application of this powerful genetic screening strategy will not only facilitate the rapid identification of genes important for bacterial toxicity, but will also enable the discovery of genes that participate in a broad range of biological processes. Given the importance to establish an effective genome-editing technology for functional genomics in a high-throughput fashion, the international competition has been fierce. Two parallel studies reported a similar methodology in Science. However, our approach is better suited for broader range of cell types, and is particularly advantageous for knowledge-based screening.

Identifying the biological roles of the newly discovered DNA modifications, including 5-formylcytosine (5fC) remains a central topic in nucleic acids epigenetics, while the major challenge is the lack of selective and sensitive sequencing methods for the genome-wide analysis. In our recent study, we presents fC-CET (cyclization-enabled C-to-T transition of 5fC), a bisulfite-free, base-resolution sequencing method for whole-genome analysis of 5fC. fC-CET involves selective chemical labeling of 5fC and subsequent C-to-T transition of labeled product during PCR amplification. With this method, we figured out the high-confident 5fC maps in mouse embryonic stem cells, and found that 5fC-marked regions are more active than 5hmC-marked ones. Since the chemical treatment of this method demonstrates no detectable DNA degradation, fC-CET also has potential for analysis of precious DNA including clinical samples (Nat Methods, 2015,12: 1047-1050).

We sequenced and assembled the tetraploid Gossypium hirsutum (AADD), diploid G. arboreum (AA) and Gossypium raimondii (DD) genomes (Nature Genet. 2012, 44: 1098-1103; 2014, 46: 567-572; Nature Biotech., 2015, 33: 524-530). Analysis of these three genome sequences revealed that two whole-genome duplications were shared by G. arboreum and Gossypium raimondii before speciation, and homeologous exchanges (HEs) taken place among cotton genomes quite commonly. Combined with the large-scale transcriptome analyses and comparative transcriptome studies, we suggest that TE-insertion-induced changes in branch point-site distribution are important for intron retention-type alternative splicing (AS), and the key role of the nucleotide binding site (NBS)-encoding gene family played in resistance to Verticillium dahliae. Our results indicate that the elongating fiber cells may expand via a linear cell-growth mode. Sequence alignment indicated that a single MYB binding site was lost in ACO1 promoter from the A genome, whereas two additional MYB binding sites were created in ACO3 promoter from the D genome. The gel shift also showed these fragment containing the MYB sites could bind with the nuclear extracts, suggesting that MYB transcription factors might play a crucial role in regulating cotton ACO gene expression.

In the future, the lab will continue to combine basic research that aims to uncover the mystery of nature with applied basic research that answers to China’s economic and developmental needs. It will build upon existing scientific expertise and technical platforms towards more significant discoveries in the areas of bio-molecules and biomedicine, plant molecular and genomic biology, plant and microbial interactions, as well as bioinformatics and genome evolution.