The genome of the colonial hydroid Hydractinia reveals that their stem cells use a toolkit of evolutionarily shared genes with all animals.

Hydractinia is a colonial marine hydroid that shows remarkable biological properties, including the capacity to regenerate its entire body throughout its lifetime, a process made possible by its adult migratory stem cells, known as i-cells. Here, we provide an in-depth characterization of the genomic structure and gene content of two Hydractinia species, Hydractinia symbiolongicarpus and Hydractinia echinata, placing them in a comparative evolutionary framework with other cnidarian genomes. We also generated and annotated a single-cell transcriptomic atlas for adult male H. symbiolongicarpus and identified cell-type markers for all major cell types, including key i-cell markers. Orthology analyses based on the markers revealed that Hydractinia's i-cells are highly enriched in genes that are widely shared amongst animals, a striking finding given that Hydractinia has a higher proportion of phylum-specific genes than any of the other 41 animals in our orthology analysis. These results indicate that Hydractinia's stem cells and early progenitor cells may use a toolkit shared with all animals, making it a promising model organism for future exploration of stem cell biology and regenerative medicine. The genomic and transcriptomic resources for Hydractinia presented here will enable further studies of their regenerative capacity, colonial morphology, and ability to distinguish self from nonself.

Natural allelic variations provide insights into host adaptation of Phytophthora avirulence effector PsAvr3c.

Filamentous pathogens, such as fungi and oomycetes, secrete avirulence (AVR) effectors that trigger plant immune responses and provide striking examples of host adaptations. Avr effector genes display different types of allelic variations, including deletions, epigenetic silencing and sequence polymorphisms, to avoid detection. However, how effector sequence polymorphisms enable pathogens to dodge host immune surveillance remains largely unknown. PsAvr3c is a Phytophthora AVR gene that is recognized by soybean carrying Rps3c. PsAvr3c natural alleles display a rich diversity of single nucleotide polymorphisms in field isolates. We combined both site-directed mutagenesis and population sequence surveys to identify a serine substitution of glycine at position 174 in PsAvr3c that resulted in evasion of Rps3c-mediated soybean immunity. The S174G substitution did not affect the nuclear localization of PsAvr3c in planta, which is required to activate Rps3c, but it significantly impaired the binding affinity of PsAvr3c with a previously identified spliceosome-associated protein GmSKRPs. Silencing GmSKRPs specifically impaired PsAvr3c-triggered cell death in Rps3c soybean. This study uncovered a plant Phytophthora pathogen that adapted to a resistant plant through a key amino acid mutation and subsequently reduced the binding affinity with a plant immune regulator to evade host resistance.

Characterization of the Papain-Like Protease p29 of the Hypovirus CHV1-CN280 in Its Natural Host Fungus Cryphonectria parasitica and Nonhost Fungus Magnaporthe oryzae.

Cryphonectria hypovirus 1 strain CN280 (CHV1-CN280) was isolated from North China and exhibited typical hypovirulence-associated traits. We previously reported that CHV1-CN280 was more aggressive and had a higher horizontal transmission ability between Cryphonectria parasitica isolates belonging to different vegetative compatibility groups than two other CHV1 hypoviruses (namely, CHV1-EP713 and CHV1-Euro7), thus displaying greater potential for biological control of chestnut blight. The genome sequence of CHV1-CN280 shared approximately 70% identity with three other hypoviruses (CHV1-EP713, CHV1-Euro7, and CHV1-EP721). The coding region for p29, a papain-like protease encoded by CHV1-CN280 hypovirus, displayed an average of only approximately 60% amino acid identity among them, while the identity between the other three CHV1 isolates was higher than 89%. Protease p29 acted as a virus-encoded determinant responsible for altering fungal host phenotypes in other CHV1 isolates. In this study, the impacts of CHV1-CN280 p29 expression in virus-free C. parasitica were investigated. CHV1-CN280 p29 expression in C. parasitica resulted in significantly reduced sporulation, pigmentation, extracellular laccase activities, and pathogenicity, which is consistent with previous investigations. Subsequently, the potential of CHV1-CN280 p29 as a viral determinant responsible for suppression of host phenotypes in other phytopathogenic fungi such as Magnaporthe oryzae, the causal agent of rice blast disease, was discussed. However, heterologous expression of p29 in M. oryzae induced the opposite effect on sporulation, extracellular laccase activities, and pathogenicity; had no significant effect on pigmentation and mycelial growth; and contributed to extracellular peroxidase activities, suggesting that CHV1-CN280 p29 may disturb a unique regulatory pathway in C. parasitica, rather than a basic regulatory pathway conserved in diverse range of fungi. Alternatively, CHV1-CN280 p29-mediated modulation of fungal phenotypes may be facilitated by the specific interaction between p29 and a special fungal-host component, which exists only with C. parasitica but not M. oryzae.

The genome of the colonial hydroid Hydractinia reveals their stem cells utilize a toolkit of evolutionarily shared genes with all animals.

Hydractinia is a colonial marine hydroid that exhibits remarkable biological properties, including the capacity to regenerate its entire body throughout its lifetime, a process made possible by its adult migratory stem cells, known as i-cells. Here, we provide an in-depth characterization of the genomic structure and gene content of two Hydractinia species, H. symbiolongicarpus and H. echinata, placing them in a comparative evolutionary framework with other cnidarian genomes. We also generated and annotated a single-cell transcriptomic atlas for adult male H. symbiolongicarpus and identified cell type markers for all major cell types, including key i-cell markers. Orthology analyses based on the markers revealed that Hydractinia's i-cells are highly enriched in genes that are widely shared amongst animals, a striking finding given that Hydractinia has a higher proportion of phylum-specific genes than any of the other 41 animals in our orthology analysis. These results indicate that Hydractinia's stem cells and early progenitor cells may use a toolkit shared with all animals, making it a promising model organism for future exploration of stem cell biology and regenerative medicine. The genomic and transcriptomic resources for Hydractinia presented here will enable further studies of their regenerative capacity, colonial morphology, and ability to distinguish self from non-self.

Gene knockdown via electroporation of short hairpin RNAs in embryos of the marine hydroid Hydractinia symbiolongicarpus.

Analyzing gene function in a broad range of research organisms is crucial for understanding the biological functions of genes and their evolution. Recent studies have shown that short hairpin RNAs (shRNAs) can induce gene-specific knockdowns in two cnidarian species. We have developed a detailed, straightforward, and scalable method to deliver shRNAs into fertilized eggs of the hydrozoan cnidarian Hydractinia symbiolongicarpus via electroporation, yielding effective gene-targeted knockdowns that can last throughout embryogenesis. Our electroporation protocol allows for the transfection of shRNAs into hundreds of fertilized H. symbiolongicarpus eggs simultaneously with minimal embryo death and no long-term harmful consequences on the developing animals. We show RT-qPCR and detailed phenotypic evidence of our method successfully inducing effective knockdowns of an exogenous gene (eGFP) and an endogenous gene (Nanos2), as well as knockdown confirmation by RT-qPCR of two other endogenous genes. We also provide visual confirmation of successful shRNA transfection inside embryos through electroporation. Our detailed protocol for electroporation of shRNAs in H. symbiolongicarpus embryos constitutes an important experimental resource for the hydrozoan community while also serving as a successful model for the development of similar methods for interrogating gene function in other marine invertebrates.

Publisher Correction: Gene knockdown via electroporation of short hairpin RNAs in embryos of the marine hydroid Hydractinia symbiolongicarpus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Manipulating Endoplasmic Reticulum-Plasma Membrane Tethering in Plants Through Fluorescent Protein Complementation.

The bimolecular fluorescence complementation (BiFC) assay has been widely used to examine interactions between integral and peripheral proteins within putative plasma membrane (PM) microdomains. In the course of using BiFC assays to examine the co-localization of plasma membrane (PM) targeted receptor-like kinases (RLKs), such as FLS2, with PM micro-domain proteins such as remorins, we unexpectedly observed heterogeneous distribution patterns of fluorescence on the PM of Nicotiana benthamiana leaf cortical cells. These patterns appeared to co-localize with the endoplasmic reticulum (ER) and with ER-PM contact sites, and closely resembled patterns caused by over-expression of the ER-PM tether protein Synaptotagmin1 (SYT1). Using domain swap experiments with SYT1, we inferred that non-specific dimerization between FLS2-VenusN and VenusC-StRem1.3 could create artificial ER-PM tether proteins analogous to SYT1. The same patterns of ER-PM tethering were produced when a representative set of integral membrane proteins were partnered in BiFC complexes with PM-targeted peripheral membrane proteins, including PtdIns(4)P-binding proteins. We inferred that spontaneous formation of mature fluorescent proteins caused the BiFC complexes to trap the integral membrane proteins in the ER during delivery to the PM, producing a PM-ER tether. This phenomenon could be a useful tool to deliberately manipulate ER-PM tethering or to test protein membrane localization. However, this study also highlights the risk of using the BiFC assay to study membrane protein interactions in plants, due to the possibility of alterations in cellular structures and membrane organization, or misinterpretation of protein-protein interactions. A number of published studies using this approach may therefore need to be revisited.

Tethering of Multi-Vesicular Bodies and the Tonoplast to the Plasma Membrane in Plants.

Tethering of the plasma membrane (PM) and many organelles to the endoplasmic reticulum (ER) for communication and lipid exchange has been widely reported. However, despite growing interest in multi-vesicular bodies (MVBs) as possible sources of exosomes, tethering of MVBs to the PM has not been reported. Here we show that MVBs and the vacuolar membrane (tonoplast) could be tethered to the PM (PM-MVB/TP tethering) by artificial protein fusions or bimolecular fluorescence complementation (BiFC) complexes that contain a peripheral membrane protein that binds the PM and also a protein that binds MVBs or the tonoplast. PM-binding proteins capable of participating in PM-MVB/TP tethering included StRem1.3, BIK1, PBS1, CPK21, and the PtdIns(4)-binding proteins FAPP1 and Osh2. MVB/TP-binding proteins capable of participating in tethering included ARA6, ARA7, RHA1, RABG3f, and the PtdIns(3)P-binding proteins Vam7p and Hrs-2xFYVE. BiFC complexes or protein fusions capable of producing PM-MVB/TP tethering were visualized as large well-defined patches of fluorescence on the PM that could displace PM proteins such as AtFlotillin1 and also could displace cytoplasmic proteins such as soluble GFP. Furthermore, we identified paralogous ubiquitin E3 ligase proteins, SAUL1 (AtPUB44), and AtPUB43 that could produce PM-MVB/TP tethering. SAUL1 and AtPUB43 could produce tethering in uninfected tissue when paired with MVB-binding proteins or when their E3 ligase domain was deleted. When Nicotiana benthamiana leaf tissue was infected with Phytophthora capsici, full length SAUL1 and AtPUB43 localized in membrane patches consistent with PM-MVB/TP tethering. Our findings define new tools for studying PM-MVB/TP tethering and its possible role in plant defense. SIGNIFICANCE STATEMENT: Although not previously observed, the tethering of multi-vesicular bodies to the plasma membrane is of interest due to the potential role of this process in producing exosomes in plants. Here we describe tools for observing and manipulating the tethering of multi-vesicular bodies and the tonoplast to the plant plasma membrane, and describe two plant proteins that may naturally regulate this process during infection.