Hybrid Screening
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2021 ◽  
Vol 12 ◽  
Shijuan Li ◽  
Bingliang Xu ◽  
Xiaolei Niu ◽  
Xiang Lu ◽  
Jianping Cheng ◽  

Agrobacterium tumefaciens can cause crown gall tumors by transferring both an oncogenic piece of DNA (T-DNA) and several effector proteins into a wide range of host plants. For the translocated effector VirE3 multiple functions have been reported. It acts as a transcription factor in the nucleus binding to the Arabidopsis thaliana pBrp TFIIB-like protein to activate the expression of VBF, an F-box protein involved in degradation of the VirE2 and VIP1 proteins, facilitating Agrobacterium-mediated transformation. Also VirE3 has been found at the plasma membrane, where it could interact with VirE2. Here, we identified AtJAZ8 in a yeast two-hybrid screening with VirE3 as a bait and confirmed the interaction by pull-down and bimolecular fluorescence complementation assays. We also found that the deletion of virE3 reduced Agrobacterium virulence in a root tumor assay. Overexpression of virE3 in Arabidopsis enhanced tumorigenesis, whereas overexpression of AtJAZ8 in Arabidopsis significantly decreased the numbers of tumors formed. Further experiments demonstrated that AtJAZ8 inhibited the activity of VirE3 as a plant transcriptional regulator, and overexpression of AtJAZ8 in Arabidopsis activated AtPR1 gene expression while it repressed the expression of AtPDF1.2. Conversely, overexpression of virE3 in Arabidopsis suppressed the expression of AtPR1 whereas activated the expression of AtPDF1.2. Our results proposed a novel mechanism of counter defense signaling pathways used by Agrobacterium, suggesting that VirE3 and JAZ8 may antagonistically modulate the salicylic acid/jasmonic acid (SA/JA)-mediated plant defense signaling response during Agrobacterium infection.

2021 ◽  
Vol 12 ◽  
Qiuping Tan ◽  
Shan Jiang ◽  
Ning Wang ◽  
Xiao Liu ◽  
Xinhao Zhang ◽  

The OVATE family protein (OFP) genes (OFPs) have been shown to respond to salt stress in plants. However, the regulatory mechanism for salt tolerance of the peach (Prunus persica) OFP gene PpOFP1 has not been elucidated. In this study, using yeast two-hybrid screening, we isolated a nucleus-localized ZF-HD_dimer domain protein PpZFHD1, which interacts with the PpOFP1 protein in the peach cultivar “Zhongnongpan No.10”. A segmentation experiment further suggested that the interaction happens more specifically between the N-terminal, contains ZF-HD_dimer domain, of PpZFHD1 and the C-terminal, consists of OVATE domain, of PpOFP1. Additionally, quantitative real-time polymerase chain reaction (qRT-PCR) experiments indicate that transcription of these two genes are induced by 200 mmol/L (mM) NaCl treatment. Heterogeneous transformation experiments suggested that the growth status of transformed yeast strain over-expressing each of these two genes was more robust than that of control (CK). Furthermore, transgenic tomato plants over-expressing PpOFP1 were also more robust. They had a higher content of chlorophyll, soluble proteins, soluble sugars, and proline. Activities of the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in these plants were higher, and tissues from these plants exhibited a lower relative conductivity and malondialdehyde (MDA) content. These results suggest that PpOFP1 physically interacts with PpZFHD1 and confers salt tolerance to tomato and yeast, thus revealing a novel mechanism for regulating salt tolerance in peach and other perennial deciduous trees.

2021 ◽  
Vol 12 ◽  
Miaoyu Song ◽  
Haomiao Wang ◽  
Zhe Wang ◽  
Hantang Huang ◽  
Shangwu Chen ◽  

The basic helix–loop–helix (bHLH) transcription factor family is the second largest transcription factor family in plants, and participates in various plant growth and development processes. A total of 118 bHLH genes were identified from fig (Ficus carica L.) by whole-genome database search. Phylogenetic analysis with Arabidopsis homologs divided them into 25 subfamilies. Most of the bHLHs in each subfamily shared a similar gene structure and conserved motifs. Seventy-two bHLHs were found expressed at fragments per kilobase per million mapped (FPKM) > 10 in the fig fruit; among them, 15 bHLHs from eight subfamilies had FPKM > 100 in at least one sample. bHLH subfamilies had different expression patterns in the female flower tissue and peel during fig fruit development. Comparing green and purple peel mutants, 13 bHLH genes had a significantly different (≥ 2-fold) expression. Light deprivation resulted in 68 significantly upregulated and 22 downregulated bHLH genes in the peel of the fruit. Sixteen bHLH genes in subfamily III were selected by three sets of transcriptomic data as candidate genes related to anthocyanin synthesis. Interaction network prediction and yeast two-hybrid screening verified the interaction between FcbHLH42 and anthocyanin synthesis-related genes. The transient expression of FcbHLH42 in tobacco led to an apparent anthocyanin accumulation. Our results confirm the first fig bHLH gene involved in fruit color development, laying the foundation for an in-depth functional study on other FcbHLH genes in fig fruit quality formation, and contributing to our understanding of the evolution of bHLH genes in other horticulturally important Ficus species.

2021 ◽  
Vol 12 ◽  
Weidong Wang ◽  
Jiajun Nie ◽  
Luqiong Lv ◽  
Wan Gong ◽  
Shuaile Wang ◽  

To successfully colonize the plants, the pathogenic microbes secrete a mass of effector proteins which manipulate host immunity. Apple valsa canker is a destructive disease caused by the weakly parasitic fungus Valsa mali. A previous study indicated that the V. mali effector protein 1 (VmEP1) is an essential virulence factor. However, the pathogenic mechanism of VmEP1 in V. mali remains poorly understood. In this study, we found that the apple (Malus domestica) pathogenesis-related 10 proteins (MdPR10) are the virulence target of VmEP1 using a yeast two-hybrid screening. By bimolecular fluorescence (BiFC) and coimmunoprecipitation (Co-IP), we confirmed that the VmEP1 interacts with MdPR10 in vivo. Silencing of MdPR10 notably enhanced the V. mali infection, and overexpression of MdPR10 markedly reduced its infection, which corroborates its positive role in plant immunity against V. mali. Furthermore, we showed that the co-expression of VmEP1 with MdPR10 compromised the MdPR10-mediated resistance to V. mali. Taken together, our results revealed a mechanism by which a V. mali effector protein suppresses the host immune responses by interfering with the MdPR10-mediated resistance to V. mali during the infection.

eLife ◽  
2021 ◽  
Vol 10 ◽  
Maria Carmela Filomena ◽  
Daniel L Yamamoto ◽  
Pierluigi Carullo ◽  
Roman Medvedev ◽  
Andrea Ghisleni ◽  

Myopalladin (MYPN) is a striated muscle-specific immunoglobulin domain-containing protein located in the sarcomeric Z-line and I-band. MYPN gene mutations are causative for dilated (DCM), hypertrophic and restrictive cardiomyopathy. In a yeast two-hybrid screening, MYPN was found to bind to titin in the Z-line, which was confirmed by microscale thermophoresis. Cardiac analyses of MYPN knockout (MKO) mice showed the development of mild cardiac dilation and systolic dysfunction, associated with decreased myofibrillar isometric tension generation and increased resting tension at longer sarcomere lengths. MKO mice exhibited a normal hypertrophic response to transaortic constriction (TAC), but rapidly developed severe cardiac dilation and systolic dysfunction, associated with fibrosis, increased fetal gene expression, higher intercalated disc fold amplitude, decreased calsequestrin-2 protein levels, and increased desmoplakin and SORBS2 protein levels. Cardiomyocyte analyses showed delayed Ca2+ release and reuptake in unstressed MKO mice as well as reduced Ca2+ spark amplitude post-TAC, suggesting that altered Ca2+ handling may contribute to the development of DCM in MKO mice.

2021 ◽  
Usman Ghani ◽  
Rahmat Ullah ◽  
Tayyaba Anjum ◽  
Yasir Ali ◽  
Muhammad Farooq Sabar ◽  

Abstract The Toll-interacting protein (TOLLIP), first detected by hybrid screening using Interleukin-1 receptor accessory protein in 2000, is ubiquitous and its TLR signaling cascade gets negatively regulated by TOLLIP in particular by impeding the TLR4 and TLR2 pathways. Toll-interacting protein facilitates TLR and TGF-β type 1 receptor intracellular localization and lysosomal degradation and exerts its anti-apoptosis and pro-autophagy effects through interaction with a target of Myb1 membrane trafficking protein 1 (TOM1) in autoimmunity. It also protects intestinal epithelial cells from apoptosis induced by (TNF-α) (IFN-γ) signaling and acts as a cargo adaptor linking (ATG8) autophagy gene 8 and microtubule-associated protein 1 light chain 3. Ubiquitin-modified cell debris along with coated autophagosomes removes harmful protein aggregates and maintains cellular homeostasis. It is therefore structurally and functionally important to detect TOLLIP polymorphisms to indicate the possible malfunctions and therapeutics. We have identified the gap of available data on nsSNPs in the TOLLIP gene in previous studies. Hence, We have used a wide range of bioinformatic techniques in this study to identify the most destructive nsSNPs in the TOLLIP gene. The in-silico tools such as PROVEAN, SIFT, SNP&GO, PhD SNP, and PolyPhen2 have been used followed by I Mutant, MutPred, and ConSurf. The 3-D mapping was carried out with I-TASSER and Phyre2. Though, STRING and GeneMANIA proposed the gene to gene interaction of TOLLIP. Our study identified G19D (rs866744102), G32R (rs1308704061), D71N (rs777772934), and E72G (rs1202660177) as the four most lethal non-synonymous SNPs in TOLLIP genes, which may play an essential part in defects of TOLLIP Protein and probably cause a different type of diseases. This is the first study of its kind, and it could pave the way in the field of medical drugs in the future. The influence of these nsSNPs in the normal functioning of TOLLIP can also be evaluated in animal models to identify the underlying biochemical pathway involved in diseases.

2021 ◽  
Liping Huang ◽  
Mohsin Tanveer ◽  
E Ji ◽  
Sergey Shabala ◽  
Mingyi Jiang

Abscisic acid (ABA) is a key component of many signaling networks mediating plant adaptation to various stresses. In this context, ABA-induced antioxidant defence is considered to be a main mechanism to that enhances water stress tolerance in plants. The specific details of this activation remain, however, elusive. In this work, we show that DIP1, a protein from novel R3H family, played a central role in modulating water stress tolerance in rice. OsDIP1 transcripts were induced by hydrogen peroxide (H2O2), ABA, drought (polyethylene glycol treatment), and salt stress. Overexpression of OsDIP1 in rice enhanced drought and salinity tolerance while knocking out OsDIP1 by CRISPR-Cas9 editing resulted in drought and salt sensitive phenotype. The activity and gene expression of antioxidant defence enzymes, superoxide dismutase (SOD), catalase (CAT), increased in OsDIP1-overexpressed transgenic rice plants, while the content of malondialdehyde (MDA) decreased. In contrast, the content and gene expression of SOD and CAT, decreased, and the content of MDA increased in knockout of OsDIP1 rice plants, suggesting that overexpression of OsDIP1 enhances the antioxidant capacity of rice plants. The yeast two hybrid screening test revealed that OsDIP1 interacted with ZFP36, a key zinc finger transcription factor involved in ABA-induced antioxidant defence. Moreover, OsDIP1 could modulate some key ABA-responsive genes via interacting with ZFP36. Overall, our findings indicate an important role of OsDIP1 in ABA-induced antioxidant defence signaling and adaptation to salinity and drought in rice.

Horticulturae ◽  
2021 ◽  
Vol 7 (9) ◽  
pp. 323
Qian Chen ◽  
Danlong Jing ◽  
Shuming Wang ◽  
Fan Xu ◽  
Chaoya Bao ◽  

NAC transcription factors (TFs) are plant-specific TFs that play essential roles in plant development; however, the function of NAC TFs in loquat development remains unknown. The natural triploid loquat (Eriobotrya japonica Lindl.), Longquan No.1. B355, has larger organs than its corresponding diploid loquat (B2). Here, we cloned an NAC-like TF (EjNACL47 (NAC-like 47)) from the cDNA of triploid loquat B355 flowers. EjNACL47 has a conserved domain of NAC TFs and is homologous to AtNAC47. Transient expression in tobacco leaves revealed that EjNACL47 localized to the nucleus, and yeast-two-hybrid screening confirmed that the C-terminus displayed transcriptional activity. Interestingly, real-time qRT-PCR indicated that the expression levels of EjNACL47 in leaves and flower organs in triploid loquat (B355) were higher than those in diploid loquat (B2), implying that EjNACL47 might be associated with the larger organ size in B355. Moreover, Arabidopsis lines ectopically expressing EjNACL47 presented obviously larger leaves, flowers, and siliques than the wild-type variant, suggesting that EjNACL47 plays a positive role in Arabidopsis organ enlargement. These results offer insight into the molecular mechanism of NAC TFs involved in regulating organ size in loquat.

2021 ◽  
Vol 11 (1) ◽  
Hong-Le Wang ◽  
Chun-Ling Xu ◽  
Chun Chen ◽  
Shan-Wen Ding ◽  
Jun-Yi Li ◽  

AbstractPlant kinases containing the LysM domain play important roles in pathogen recognition and self-defense reactions. And it could recognize microbe-associated molecules including chitin and other polypeptides. The white tip nematode Aphelenchoides besseyi is a migratory parasitic nematode that infects plant shoots. It is distributed over almost all rice-producing areas and causes up to 50% economic losses. The rice OsRLK3 gene was a defense-related LysM kinase gene of rice. This study showed that the rice LysM kinase OsRLK3 could be induced by flg22, jasmonic acid, salicylic acid, and chitin. An interaction gene, Ab-atps from A. besseyi, was identified by screening the interaction between the rice gene OsRLK3 and an A. besseyi cDNA library using yeast two-hybrid screening. Ab-atps is a novel ATP synthase gene with a full length of 1341 bp, coding for 183 amino acids. The mRNA of Ab-atps was located in the esophagus and reproductive system of A. besseyi. The expression of Ab-atps was assessed at different developmental stages of the nematode and found to be the highest in the juvenile, followed by the egg, female, and male. Reproduction was significantly decreased in nematodes treated with Ab-atps double-stranded RNA (dsRNA) (p < 0.05). Transient expression experiments showed that Ab-ATPS-GFP was distributed in the nucleus, cytoplasm, and cell membrane, and Ab-ATPS-GFP triggered plant cell death. OsRLK3 was expressed significantly higher at 0.5 day and 1 day (p < 0.05) in rice plants inoculated with nematodes treated with Ab-atps dsRNA and gfp dsRNA for 0.5–7 days, respectively. Further, OsRLK3 expression under Ab-atps dsRNA treatment was significantly lower than with gfp dsRNA treatment at 0.5 day (p < 0.05) and significantly higher than with gfp dsRNA treatment at 1 day (p < 0.05). These results suggest that rice OsRLK3 could interact with A. besseyi Ab-atps, which plays an important role in growth, reproduction, and infection of the nematode. Our findings provide a theoretical basis to further understand the parasitic strategy of A. besseyi and its interaction mechanism with host plants, suggesting new ideas and targets for controlling A. besseyi.

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