July 2 at ICAR (morning sessions)

2 July ICAR 2016

This morning I attended the Peptide Hormone session and the Genome Editing session – both were excellent. I hope I’ve captured them adequately – be sure to have a look at the referenced papers for more info than I was able to provide in this “on the fly” report!

Session 17 (Hormone – Peptide)

Yoshikatsu Matsubayashi (Nagoya University, Japan) – Identification of novel peptide ligand-receptor pairs in plants

22 years ago when Yoshikatsu first presented on peptide hormone, there was no peptide hormone category, so he presented in “other compounds”, so he’s delighted to have a dedicated session on peptide hormones. Two key words for today, “protein-based approach” and “insights from species other than Arabidopsis”

Arabidopsis has 1000 secreted peptides, 600 receptor kinases. The number of known peptide ligand – receptor pairs is about 30, so we can anticipate many additional pairs to be found. Examples: PSK, CLV3  Background 2014 Annu Rev Plant Biol.

Approach: in silico screening, structural determination of mature peptides, finally, analysis of receptor interaction. What do you look for to find a peptide hormone? Secretion signal sequence, diversified sequence, conserved sequence. Look for presence as a family, ORF about 100 amino acids, conserved region at C term.

See Identification of a biologically active, small, secreted peptide in Arabidopsis by in silico gene screening, followed by LC-MS-based structure analysis Plant J 2008.  What do receptors look like? Shiu and Bleeker 2001 – RLKs in plants. In general, direct ligand-binding receptors >400 aa, co-receptors <400 aa. Express candidate receptors in BY cells, conjugate photoactivatable cross linker to putative ligand, look for interaction. In this way, identified CEPR1 and 2 – it is involved in N uptake or assimilation pathways. CEP is involved in systemic nitrogen signalling – produced in root in low N, travels to shoot, interacts with receptor, sends second signal to root to promote N uptake.  See 2014 Science paper  Perception of root-derived peptides by shoot LRR-RKs mediates systemic N-demand signaling. They are looking for downstream signal.

See also 2016 PNAS Identification of three LRR-RKs involved in perception of root meristem growth factor in Arabidopsis. RGFR involved in root growth. Expressed in root tip, required for proximal meristem development. Define expression of PLETHORA transcription factors. Another group identified SKM2 as putative receptor for RGF.

David Jackson (Cold Spring Harbor Laboratory, U.S.A) – CLE peptide signaling from primordia controls stem cells in maize and Arabidopsis shoots

David’s latest paper: Signaling from maize organ primordia via FASCIATED EAR3 regulates stem cell proliferation and yield traits. Nature Genetics, 2016. David starts with the organization and signalling within the meristem, including the CLAVATA WUSCHEL feedback loop required for shoot stems cell population (meristem) size. David’s group identified fascinated ear (fea) mutants in maize. Some of their genes are published including orthologues of CLV1 and CLV2. Today’s story concerns fea3, which provides a different twist on the CLV story. FEA3 encodes a receptor like kinase. Double mutants highly synergistic suggesting it operates in a separate pathway. Also, expression of FEA3 is lower down in meristem than previously shown for CLV genes. Also, in clv mutants, WUS expression moves UP, in fea3 mutants, WUS expression moves DOWN. Decided to see if FEA3 is a receptor for a CLE peptide, found ZmFCP1 CLE peptide gene, which is expressed in organ primordia, not SAM (different from typical CLE expression pattern). Updated model – signal moves from primordium to suppress WUS expression from below. Note that in animals and plants, stem cells are maintained by short-range communication in niches. Hsu and Fuchs, Cell 2011 for an example in animals.

Does FEA3 have a function in Arabidopsis Three orthologues, one has  no public knockout allele, but by RNAi found that suppression causes fasciation (same phenotype as in maize). A different CLE peptide binds it, which is also suppressed in peripheral zone.

Finally, note that over time the size of the maize inflorescence has increased due to selection, leading to more kernels. By introducing weak alleles of FEA3, the meristem size increased and the yield did too. Potential application in the field?

Jijie Chai (Tsinghua University, China) -Talking about research newly published in Cell Research on the structural analysis of LRR-RKs and their peptide ligands: Signature motif-guided identification of receptors for peptide hormones essential for root meristem growth and Crystal structure of PXY-TDIF complex reveals a conserved recognition mechanism among CLE peptide-receptor pairs.

Take home message: “Our data provide a structural template for understanding the recognition mechanism of CLE peptides by their receptors, offering an opportunity for the identification of receptors of other uncharacterized CLE peptides.”

Masahiro Kanaoka (Nagoya University, Japan) in the group of Tetsuya Higashiyama – Regulation of pollen tube guidance by secreted peptides and molecules

Masahiro works on signalling during pollen tube growth – see recent review in COPB Peptide signaling in pollen tube guidance. Pollen tube guidance is a key event for fertilization. He shows a video of a pollen tube tracking a secreted signal. Secreted peptides are required for pollen tube guidance – see 2009 paper Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells. The group recently identified receptors on the pollen tube tip that control LURE sensing -> Tip-localized receptors control pollen tube growth and LURE sensing in Arabidopsis.

Their group also recently identified an arabinogalactan required for successful fertilization.  The AMOR Arabinogalactan Sugar Chain Induces Pollen-Tube Competency to Respond to Ovular Guidance.

Session 18 (Plant Lipids)

John Browse (Washington State University, U.S.A) – Arabidopsis Reveals Lipid metabolism in Oilseeds

Mi Chung Suh (Chonnam National University, Korea) – AP2/ERF-type transcription factors contribute to the regulation of organ-specific cuticular wax biosynthesis in Arabidopsis

Edgar B. Cahoon (University of Nebraska, U.S.A) – To Grow or Die: Unraveling the Sphingolipid Homeostatic Regulatory Network in Plants

Yuki Nakamura (Academia Sinica, Taiwan) – Phosphatidylcholine in Arabidopsis: Biosynthesis and function in plant development

11:00 – 12:30     Session 19 (Systems Biology)

Barry Pogson (Australian National University, Australia) – Abiotic Stress Memory: Learning to Forget

Siobhan Brady (UC-Davis, U.S.A) – Transcriptional regulation of plant metabolism

Meike Burow (University of Copenhagen, Denmark) – Natural variation in networks linking glucosinolates to growth and flowering time

Olivier Van Akan (University of Western Australia, Australia) – Mitochondrial and chloroplast stress responses are modulated in distinct touch and chemical inhibition phases in Arabidopsis

Session 20 (Genome Editing)

Jinsoo Kim (Institute for Basic Science, Korea) – CRISPR RNA-guided Genome Editing in Human Stem Cells, Animals, and Plants. 2014 review A guide to genome engineering with programmable nucleases.

Many genetic diseases are caused by genome inversion, including hemophelia.  Haemophilia mice have been corrected with gene-corrected cells.  Woo and Kim Nat Biotechnol 2015 RNA guided genome editing in lettuce. No exogenous DNA used, (shouldn’t require GMO regulations). Possible to do whole genome sequencing (Digenome-seq) that profiles sites where off-target double-strand genome cuts were produced. Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq. Cleavage sites were validated. Only five of these additional off-target sites were mutated. Modified sgRNAs reduce off-target effects – add two extra G at end to increase specificity. Many advantages to Digenome-Seq (see paper).

Cas9 variants or other enzymes can be used to decrease off-target sites – for example Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. See this website for webtools http://www.rgenome.net/.

Caixia Gao (Chinese Academy of Sciences, China) – Precision plant breeding using genome editing technologies (note that Caixia Gao was recently highlighted as a science star in Nature). See Precision Genome Engineering and Agriculture: Opportunities and Regulatory Challenges. See Genome editing in rice and wheat using the CRISPR/Cas system.

Crop breeding – potential to avoid regulatory issues by using genome editing (see for example http://onlinelibrary.wiley.com/doi/10.1038/embor.2012.168/abstract).

Examples of the use of genome editing in crops: Wheat rescued from fungal disease, Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew (using TALENs). Another study: Establishing a CRISPR–Cas-like immune system conferring DNA virus resistance in plants.

Nicola J. Patron (Earlham Institute, formerly The Genome Analysis Centre, U.K) – The Application of Synthetic Biology to Plant Genome Engineering. All of our electronic gadgets are compatible. Screws are compatible. Can biological materials be standardized? Biobricks. Standards for plant synthetic biology: a common syntax for exchange of DNA parts.  See the library of parts in addgene. See Multi-gene engineering in plants with RNA-guided Cas9 nuclease.  Here’s a paper that shows this being used in plants: Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease. She also presented several projects in progress involving genome editing in tomato and potato and others. Finally she introduced digital PCR – divide your PCR reaction into 20,000 droplets, get an accurate quantification of sample. She didn’t provide a reference but several company websites describe this method.

Christopher Town (J. Craig Venter Institute, U.S.A) – Araport – A Community Platform for Data Sharing, Integration, and Discovery

White paper announcing the project Taking the Next Step: Building an Arabidopsis Information Portal  Effort to collect all data, much of it moves into Araport in real time.

A very visual presentation as he walked us through the windows and functions – play with it yourself. Thalemine (data warehouse) Data types: chromosomes, transcripts, proteins, expression, polymorphisms, your own data. Jbrowse (genome browser). App store. Note that they are finishing a re-annotation of the genome, starting with TAIR10 and moving to Araport11. Many significant changes in the new annotation.

Take home message – Use it. Also, make sure funders understand value of Arabidopsis research.

 

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