TRANSGENIC SOYBEAN PLANTS EXPRESSING ASOYBEAN HOMOLOG OF GLYCINE-RICH PROTEIN 7 (GRP7) AND EXHIBITING IMPROVED INNATE IMMUNITY

This disclosure provides for transgenic Soybean plants expressing a soybean homolog of glycine-rich protein 7 (GRP7) and exhibiting improved innate immunity and meth ods of making Such plants

TRANSGENIC SOYBEAN PLANTS EXPRESSING ASOYBEAN HOMOLOG OF GLYCINE-RICH PROTEIN 7 (GRP7) AND EXHIBITING IMPROVED INNATE IMMUNITY

This disclosure provides for transgenic Soybean plants expressing a soybean homolog of glycine-rich protein 7 (GRP7) and exhibiting improved innate immunity and meth ods of making Such plants

\u3ci\u3ePseudomonas\u3c/i\u3e HopU1 modulates plant immune receptor levels by blocking the interaction of their mRNAs with GRP7

Pathogens target important components of host immunity to cause disease. The Pseudomonas syringae type III-secreted effector HopU1 is a mono-ADP-ribosyltransferase required for full virulence on Arabidopsis thaliana. HopU1 targets several RNA-binding proteins including GRP7, whose role in immunity is still unclear. Here, we show that GRP7 associates with translational components, as well as with the pattern recognition receptors FLS2 and EFR. Moreover, GRP7 binds specifically FLS2 and EFR transcripts in vivo through its RNA recognition motif. HopU1 does not affect the protein–protein associations between GRP7, FLS2 and translational components. Instead, HopU1 blocks the interaction between GRP7 and FLS2 and EFR transcripts in vivo. This inhibition correlates with reduced FLS2 protein levels upon Pseudomonas infection in a HopU1- dependent manner. Our results reveal a novel virulence strategy used by a microbial effector to interfere with host immunity

Structure Function Analysis of an ADP-ribosyltransferase Type III Effector and Its RNA-binding Target in Plant Immunity

Background: HopU1 ADP-ribosylates GRP7, suppressing plant immunity. Results: The HopU1 structure has two novel loops required for GRP7 recognition, and HopU1 ribosylates GRP7 at an arginine in position 49 disrupting its function. Conclusion: HopU1 targets a conserved arginine in GRP7, disabling its ability to bind immunity-related RNA. Significance: The mechanistic details of how HopU1 recognizes its substrate reveal how HopU1 contributes to pathogenesis

Structure Function Analysis of an ADP-ribosyltransferase Type III Effector and Its RNA-binding Target in Plant Immunity

Background: HopU1 ADP-ribosylates GRP7, suppressing plant immunity. Results: The HopU1 structure has two novel loops required for GRP7 recognition, and HopU1 ribosylates GRP7 at an arginine in position 49 disrupting its function. Conclusion: HopU1 targets a conserved arginine in GRP7, disabling its ability to bind immunity-related RNA. Significance: The mechanistic details of how HopU1 recognizes its substrate reveal how HopU1 contributes to pathogenesis

Structure Function Analysis of an ADP-ribosyltransferase Type III Effector and Its RNA-binding Target in Plant Immunity

Background: HopU1 ADP-ribosylates GRP7, suppressing plant immunity. Results: The HopU1 structure has two novel loops required for GRP7 recognition, and HopU1 ribosylates GRP7 at an arginine in position 49 disrupting its function. Conclusion: HopU1 targets a conserved arginine in GRP7, disabling its ability to bind immunity-related RNA. Significance: The mechanistic details of how HopU1 recognizes its substrate reveal how HopU1 contributes to pathogenesis

\u3ci\u3ePseudomonas\u3c/i\u3e HopU1 modulates plant immune receptor levels by blocking the interaction of their mRNAs with GRP7

Pathogens target important components of host immunity to cause disease. The Pseudomonas syringae type III-secreted effector HopU1 is a mono-ADP-ribosyltransferase required for full virulence on Arabidopsis thaliana. HopU1 targets several RNA-binding proteins including GRP7, whose role in immunity is still unclear. Here, we show that GRP7 associates with translational components, as well as with the pattern recognition receptors FLS2 and EFR. Moreover, GRP7 binds specifically FLS2 and EFR transcripts in vivo through its RNA recognition motif. HopU1 does not affect the protein–protein associations between GRP7, FLS2 and translational components. Instead, HopU1 blocks the interaction between GRP7 and FLS2 and EFR transcripts in vivo. This inhibition correlates with reduced FLS2 protein levels upon Pseudomonas infection in a HopU1- dependent manner. Our results reveal a novel virulence strategy used by a microbial effector to interfere with host immunity

CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii

Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9. We obtained CRISPR/Cas9-induced mutations at three loci including MAA7, CpSRP43 and ChlM, and targeted mutagenic efficiency was improved up to 100 fold compared to the first report of transgenic Cas9-induced mutagenesis. Interestingly, we found that unrelated vectors used for the selection purpose were predominantly integrated at the Cas9 cut site, indicative of NHEJ-mediated knock-in events. As expected with Cas9 RNPs, no off-targeting was found in one of the mutagenic screens. In conclusion, we improved the knockout efficiency by using Cas9 RNPs, which opens great opportunities not only for biological research but also industrial applications in Chlamydomonas and other microalgae. Findings of the NHEJ-mediated knock-in events will allow applications of the CRISPR/Cas9 system in microalgae, including safe harboring techniques shown in other organisms.

CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii

Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9. We obtained CRISPR/Cas9-induced mutations at three loci including MAA7, CpSRP43 and ChlM, and targeted mutagenic efficiency was improved up to 100 fold compared to the first report of transgenic Cas9-induced mutagenesis. Interestingly, we found that unrelated vectors used for the selection purpose were predominantly integrated at the Cas9 cut site, indicative of NHEJ-mediated knock-in events. As expected with Cas9 RNPs, no off-targeting was found in one of the mutagenic screens. In conclusion, we improved the knockout efficiency by using Cas9 RNPs, which opens great opportunities not only for biological research but also industrial applications in Chlamydomonas and other microalgae. Findings of the NHEJ-mediated knock-in events will allow applications of the CRISPR/Cas9 system in microalgae, including "safe harboring" techniques shown in other organisms142561sciescopu

Transgene and Transposon Silencing in \u3ci\u3eChlamydomonas reinhardtii\u3c/i\u3e by a DEAH-Box RNA Helicase

The molecular mechanism(s) responsible for posttranscriptional gene silencing and RNA interference remain poorly understood. We have cloned a gene (Mut6) from the unicellular green alga Chlamydomonas reinhardtii that is required for the silencing of a transgene and two transposon families. Mut6 encodes a protein that is highly homologous to RNA helicases of the DEAH-box family. This protein is necessary for the degradation of certain aberrant RNAs, such as improperly processed transcripts, which are often produced by transposons and some transgenes