A Protein-trap allele reveals roles for Drosophila ATF4 in photoreceptor degeneration, oogenesis and wing development

Metazoans have evolved various quality control mechanisms to cope with cellular stress inflicted by external and physiological conditions. ATF4 is a major effector of the Integrated Stress Response (ISR), an evolutionarily conserved pathway that mediates adaptation to various cellular stressors. Loss of function of Drosophila ATF4, encoded by the gene cryptocephal (crc), results in lethality during pupal development. The roles of crc in Drosophila disease models and in adult tissue homeostasis thus remain poorly understood. Here, we report that a protein-trap MiMIC insertion in the crc locus generates a crc-GFP fusion protein that allows visualization of crc activity in vivo. This allele also acts as a hypomorphic mutant that uncovers previously unknown roles for crc. Specifically, the crc protein-trap line shows crc-GFP induction in a Drosophila model for Retinitis Pigmentosa (RP). This crc allele renders flies more vulnerable to amino acid deprivation and age-dependent retinal degeneration. These mutants also show defects in wing veins and oocyte maturation. Together, our data reveal previously unknown roles for crc in development, cellular homeostasis and photoreceptor survival.

Heterologous expression of the human ZIP4 zinc transporter in Saccharomyces cerevisiae

The human (h) transporter, hZIP4 is the primary zinc importer in the intestine and is also expressed in a variety of organs such as the pancreas and brain. Dysfunction of hZIP4 can result in the zinc deficiency disease acrodermatitis enteropathica (AE), which disrupts digestive and immune system homeostasis. Structure-function studies of hZIP4 have been greatly hindered by the absence of a robust heterologous expression system. Here, we report the heterologous expression of hZIP4 in Saccharomyces cerevisiae. Both a wild type and a mutant S. cerevisiae strain, in which the endogenous zinc transporters are deleted, were used to test the expression and localization of an hZIP4-GFP fusion protein. A full-length hZIP4-GFP and a truncated membrane domain only (mhZIP4-GFP) protein were successfully produced and targeted to the plasma membrane in yeast.

Cystin genetic variants cause autosomal recessive polycystic kidney disease associated with altered Myc expression

Mutation of the Cys1 gene underlies the renal cystic disease in the Cys1cpk/cpk (cpk) mouse that phenocopies human autosomal recessive polycystic kidney disease (ARPKD). Cystin, the protein product of Cys1, is expressed in the primary apical cilia of renal ductal epithelial cells. In previous studies, we showed that cystin regulates Myc expression via interaction with the tumor suppressor, necdin. Here, we demonstrate rescue of the cpk renal phenotype by kidney-specific expression of a cystin-GFP fusion protein encoded by a transgene integrated into the Rosa26 locus. In addition, we show that expression of the cystin-GFP fusion protein in collecting duct cells down-regulates expression of Myc in cpk kidneys. Finally, we report the first human patient with an ARPKD phenotype due to homozygosity for a deleterious splicing variant in CYS1. These findings suggest that mutations in Cys1/CYS1 cause an ARPKD phenotype in mouse and human, respectively, and that the renal cystic phenotype in the mouse is driven by overexpression of the Myc proto-oncogene.

SUMO Modification of OsFKBP20-1b Is Integral to Proper Pre-mRNA Splicing upon Heat Stress in Rice

OsFKBP20-1b, a plant-specific cyclophilin protein, has been implicated to regulate pre-mRNA splicing under stress conditions in rice. Here, we demonstrated that OsFKBP20-1b is SUMOylated in a reconstituted SUMOylation system in E.coli and in planta, and that the SUMOylation-coupled regulation was associated with enhanced protein stability using a less SUMOylated OsFKBP20-1b mutant (5KR_OsFKBP20-1b). Furthermore, OsFKBP20-1b directly interacted with OsSUMO1 and OsSUMO2 in the nucleus and cytoplasm, whereas the less SUMOylated 5KR_OsFKBP20-1b mutant had an impaired interaction with OsSUMO1 and 2 in the cytoplasm but not in the nucleus. Under heat stress, the abundance of an OsFKBP20-1b-GFP fusion protein was substantially increased in the nuclear speckles and cytoplasmic foci, whereas the heat-responsiveness was remarkably diminished in the presence of the less SUMOylated 5KR_OsFKBP20-1b-GFP mutant. The accumulation of endogenous SUMOylated OsFKBP20-1b was enhanced by heat stress in planta. Moreover, 5KR_OsFKBP20-1b was not sufficiently associated with the U snRNAs in the nucleus as a spliceosome component. A protoplast transfection assay indicated that the low SUMOylation level of 5KR_OsFKBP20-1b led to inaccurate alternative splicing and transcription under heat stress. Thus, our results suggest that OsFKBP20-1b is post-translationally regulated by SUMOylation, and the modification is crucial for proper RNA processing in response to heat stress in rice.

Potato NAC Transcription Factor StNAC053 Enhances Salt and Drought Tolerance in Transgenic Arabidopsis

The NAC (NAM, ATAF1/2, and CUC2) transcription factors comprise one of the largest transcription factor families in plants and play important roles in stress responses. However, little is known about the functions of potato NAC family members. Here we report the cloning of a potato NAC transcription factor gene StNAC053, which was significantly upregulated after salt, drought, and abscisic acid treatments. Furthermore, the StNAC053-GFP fusion protein was found to be located in the nucleus and had a C-terminal transactivation domain, implying that StNAC053 may function as a transcriptional activator in potato. Notably, Arabidopsis plants overexpressing StNAC053 displayed lower seed germination rates compared to wild-type under exogenous ABA treatment. In addition, the StNAC053 overexpression Arabidopsis lines displayed significantly increased tolerance to salt and drought stress treatments. Moreover, the StNAC053-OE lines were found to have higher activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) under multiple stress treatments. Interestingly, the expression levels of several stress-related genes including COR15A,DREB1A, ERD11, RAB18, ERF5, and KAT2, were significantly upregulated in these StNAC053-overexpressing lines. Taken together, overexpression of the stress-inducible StNAC053 gene could enhance the tolerances to both salt and drought stress treatments in Arabidopsis, likely by upregulating stress-related genes.

Wild Soybean Oxalyl-CoA Synthetase Degrades Oxalate and Affects the Tolerance to Cadmium and Aluminum Stresses

Acyl activating enzyme 3 (AAE3) was identified as being involved in the acetylation pathway of oxalate degradation, which regulates the responses to biotic and abiotic stresses in various higher plants. Here, we investigated the role of Glycine sojaAAE3 (GsAAE3) in Cadmium (Cd) and Aluminum (Al) tolerances. The recombinant GsAAE3 protein showed high activity toward oxalate, with a Km of 105.10 ± 12.30 μM and Vmax of 12.64 ± 0.34 μmol min−1 mg−1 protein, suggesting that it functions as an oxalyl–CoA synthetase. The expression of a GsAAE3–green fluorescent protein (GFP) fusion protein in tobacco leaves did not reveal a specific subcellular localization pattern of GsAAE3. An analysis of the GsAAE3 expression pattern revealed an increase in GsAAE3 expression in response to Cd and Al stresses, and it is mainly expressed in root tips. Furthermore, oxalate accumulation induced by Cd and Al contributes to the inhibition of root growth in wild soybean. Importantly, GsAAE3 overexpression increases Cd and Al tolerances in A. thaliana and soybean hairy roots, which is associated with a decrease in oxalate accumulation. Taken together, our data provide evidence that the GsAAE3-encoded protein plays an important role in coping with Cd and Al stresses.

VAV1-GFP Fusion Protein Generated By Intron-Based Genome Editing through CRISPR/Cas9 Leads to Spontaneous Activation in TCR Signaling

Recurrent VAV1 mutations and gene fusions (VAV1-THAP4, VAV1-MYO1F, and VAV1-S100A7) have been identified in peripheral T-cell lymphoma (PTCL) including angioimmunoblastic T-cell lymphoma (AITL) patients. A common theme of these genetic aberrations is the loss of the auto-inhibitory C-terminal SH3 domain of VAV1 resulting in aberrant activation of VAV1 independent of normal activation events. Although mouse models support VAV1 mutation/fusion as having a driver oncogenic role in the pathogenesis of PTCL, investigations on VAV1 activity in human cells were performed mainly on the Jurkat cell line with exogenous expression of VAV1 fusion proteins. This approach has un-physiological expression of VAV1 and the functions of VAV1 fusion/mutation under normal endogenous regulation need to be explored. In this study, we introduced a fusion gene, similar to what has been observed in PTCL, into the endogenous VAV1 locus. The fusion gene was under normal regulatory controls instead of being over-expressed by a viral vector, thus providing a more accurate assessment of its function in vivo. To simulate VAV1 fusion, we knocked in a green fluorescence protein (GFP) sequence followed by a simian virus 40 (SV40) poly(A) signal into intron 25 of VAV1 locus by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology. A homologous DNA repair (HDR) template with tandem homologous sequences of VAV1 gene, GFP gene, and a SV40 transcription poly(A) signal was electroporated into Jurkat cells together with the Cas9/sgRNA ribonucleoprotein (RNP) complex. This knock-in disrupted the transcription of exon 26 and exon 27, resulting in an in-frame fusion protein with GFP fused to the C-terminal of SH2 of VAV1 (VAV1SH2-GFP)(Figure A). Because our guide RNA targeted the intron 25 sequence by CRISPR/Cas9 system, any possible indels caused by non-homologous end joining will occur within the intron and will not change the protein sequence of the wild type VAV1. The GFP expressing cells were isolated from the edited cell population by FACS. The fusion of GFP with VAV1 in the sorted cells was confirmed by western blot (Figure B) and these cells displayed a heterozygous VAV1SH2-GFP fusion/wild type (WT) phenotype that mimicked the VAV1 translocations observed in PTCL patients. Jurkat cells with VAV1SH2-GFP showed spontaneous activation of the T-cell receptor (TCR) signaling pathway. Analysis of signaling events downstream of VAV1 demonstrated increased phosphorylation in ITK, LCK, and subsequent ERK in Jurkat cells with VAV1SH2-GFP compared with WT Jurkat cells by western blot (Figure B). We also observed consistently elevated pERK in Jurkat cells with VAV1SH2-GFP by flow cytometry (Figure C). Notably, this elevation in pERK was spontaneous and independent of TCR stimulation with anti-CD3 antibody. VAV1SH2-GFP fusion protein also led to marked activation of downstream NFAT and NF-κB pathways as shown by Luciferase reporter assays (Figure D). Similarly, the enhanced NFAT and NF-κB pathway activation in Jurkat with the fusion protein was independent of TCR stimulation. Interestingly, with antiCD3 stimulation, Jurkat cells with VAV1SH2-GFP showed significantly lower pERK, NFAT and NF-κB activity compared to WT Jurkat cells with anti-CD3 stimulation. In conclusion, in Jurkat cells genetically edited with VAV1SH2-GFP, spontaneous activation of TCR signaling and subsequently increased NFAT and NF-κB activity were observed. Our findings further support that VAV1 C-terminal SH3 domain plays an important regulatory role in blocking VAV1 activity in the absence of proper activation. Removing C-terminal SH3 domain or replacing it with GFP or other protein relieves this inhibition, allowing spontaneous activation independent of TCR stimulation. Our study also indicates the sensitivity of the TCR signaling pathway to the level of activation and hyperactivation is detrimental. To validate the function of VAV1SH2-GFP in normal T cells, we have also successfully edited primary CD4+ T cells with VAV1SH2-GFP and the TCR signaling pathway in edited primary CD4+ T cell is currently being evaluated with and without anti-CD3/CD28 stimulation. Figure Disclosures No relevant conflicts of interest to declare.

The Glycine- and Proline-Rich Protein AtGPRP3 Negatively Regulates Plant Growth in Arabidopsis

Glycine- and proline-rich proteins (GPRPs) comprise a small conserved family that is widely distributed in the plant kingdom. GPRPs are relatively short peptides (<200 amino acids) that contain three typical domains, including an N-terminal XYPP-repeat domain, a middle hydrophobic domain rich in alanine, and a C-terminal HGK-repeat domain. These proteins have been proposed to play fundamental roles in plant growth and environmental adaptation, but their functions remain unknown. In this study, we selected an Arabidopsis GPRP (AtGPRP3) to profile the physiological role of GPRPs. Transcripts of AtGPRP3 could be detected in the whole Arabidopsis plant, but greater amounts were found in the rosette, followed by the cauline. The AtGPRP3::GFP fusion protein was mainly localized in the nucleus. The overexpression and knockout of AtGPRP3, respectively, retarded and accelerated the growth of Arabidopsis seedlings, while the increase in the growth rate of atgprp3 plants was offset by the complementary expression of AtGPRP3. CAT2 and CAT3, but not CAT1, interacted with AtGPRP3 in the nuclei of Arabidopsis protoplasts. The knockout of CAT2 by CRISPR-Cas9 retarded the growth of the Arabidopsis seedlings. Together, our data suggest that AtGPRP3 negatively regulates plant growth, potentially through CAT2 and CAT3.

A putative effector UvHrip1 inhibits BAX-triggered cell death in Nicotiana benthamiana, and infection of Ustilaginoidea virens suppresses defense-related genes expression

Rice false smut (RFS), caused by Ustilaginoidea virens, is one of the most detrimental rice fungal diseases and pose a severe threat to rice production and quality. Effectors in U. virens often act as a set of essential virulence factors that play crucial roles in the interaction between host and the pathogen. Thus, the functions of each effector in U. virens need to be further explored. Here, we performed multiple alignment analysis and demonstrated a small secreted hypersensitive response-inducing protein (hrip), named UvHrip1, was highly conserved in fungi. The predicted SP of UvHrip1 was functional, which guided SUC secreted from yeast and was recognized by plant cells. The localization of UvHrip1 was mainly in the nucleus and cytoplasm monitored through the GFP fusion protein in Nicotiana benthamiana cells. uvhrip1 was drastically up-regulated in the susceptible cultivar LYP9 of rice during the pathogen infection, while did not in the resistant cultivar IR28. We also proved that UvHrip1 suppressed the mammalian BAX-induced necrosis-like defense symptoms in N. benthamiana. Furthermore, patterns of expression of defense-related genes, OsPR1#012 and OsPR10b, were regulated over U. virens infection in rice. Collectively, our data demonstrated that infection of U. virens suppresses defense-related genes expression and UvHrip1 was most likely a core effector in regulating plant immunity.