Yeast Overexpressing Research Articles

Unravelling bacterial virulence factors in yeast: From identification to the elucidation of their mechanisms of action.

Pathogenic bacteria employ virulence factors (VF) to establish infection and cause disease in their host. Yeasts, Saccharomyces cerevisiae and Saccharomyces pombe, are useful model organisms to study the functions of bacterial VFs and their interaction with targeted cellular processes because yeast processes and organelle structures are highly conserved and similar to higher eukaryotes. In this review, we describe the principles and applications of the yeast model for the identification and functional characterisation of bacterial VFs to investigate bacterial pathogenesis. The growth inhibition phenotype caused by the heterologous expression of bacterial VFs in yeast is commonly used to identify candidate VFs. Then, subcellular localisation patterns of bacterial VFs can provide further clues about their target molecules and functions during infection. Yeast knockout and overexpression libraries are also used to investigate VF interactions with conserved eukaryotic cell structures (e.g., cytoskeleton and plasma membrane), and cellular processes (e.g., vesicle trafficking, signalling pathways, and programmed cell death). In addition, the yeast growth inhibition phenotype is also useful for screening new drug leads that target and inhibit bacterial VFs. This review provides an updated overview of new tools, principles and applications to study bacterial VFs in yeast.

SIRT1, resveratrol and aging.

Aging is linked to a time-associated decline in both cellular function and repair capacity leading to malfunction on an organismal level, increased frailty, higher incidence of diseases, and death. As the population grows older, there is a need to reveal mechanisms associated with aging that could spearhead treatments to postpone the onset of age-associated decline, extend both healthspan and lifespan. One possibility is targeting the sirtuin SIRT1, the founding member of the sirtuin family, a highly conserved family of histone deacetylases that have been linked to metabolism, stress response, protein synthesis, genomic instability, neurodegeneration, DNA damage repair, and inflammation. Importantly, sirtuins have also been implicated to promote health and lifespan extension, while their dysregulation has been linked to cancer, neurological processes, and heart disorders. SIRT1 is one of seven members of sirtuin family; each requiring nicotinamide adenine dinucleotide (NAD+) as co-substrate for their catalytic activity. Overexpression of yeast, worm, fly, and mice SIRT1 homologs extend lifespan in each animal, respectively. Moreover, lifespan extension due to calorie restriction are associated with increased sirtuin activity. These findings led to the search for a calorie restriction mimetic, which revealed the compound resveratrol; (3, 5, 4'-trihydroxy-trans-stilbene) belonging to the stilbenoids group of polyphenols. Following this finding, resveratrol and other sirtuin-activating compounds have been extensively studied for their ability to affect health and lifespan in a variety of species, including humans via clinical studies.

Alternative Splicing Plays a Crucial Role in the Salt Tolerance of Foxtail Millet.

Foxtail millet is an important cereal crop that is relatively sensitive to salt stress, with its yield significantly affected by such stress. Alternative splicing (AS) widely affects plant growth, development, and adaptability to stressful environments. Through RNA-seq analysis of foxtail millet under different salt treatment periods, 2078 AS events were identified, and analyses were conducted on differential gene (DEG), differential alternative splicing gene (DASG), and overlapping gene. To investigate the regulatory mechanism of AS in response to salt stress in foxtail millet, the foxtail millet AS genes SiCYP19, with two AS variants (SiCYP19-a and SiCYP19-b), were identified and cloned. Yeast overexpression experiments indicated that SiCYP19 may be linked to the response to salt stress. Subsequently, we conducted overexpression experiments of both alternative splicing variants in foxtail millet roots to validate them experimentally. The results showed that, under salt stress, both SiCYP19-a and SiCYP19-b jointly regulated the salt tolerance of foxtail millet. Specifically, overexpression of SiCYP19-b significantly increased the proline content and reduced the accumulation of reactive oxygen species (ROS) in foxtail millet, compared to that in SiCYP19-a. This shows that SiCYP19-b plays an important role in increasing the content of proline and promoting the clearance of ROS, thus improving the salt tolerance of foxtail millet.

HgS2, a novel salt-responsive gene from the Halophyte Halogeton glomeratus, confers salt tolerance in transgenic Arabidopsis.

Halophyte Halogeton glomeratus mostly grows in saline desert areas in arid and semi-arid regions and is able to adapt to adverse conditions such as salinity and drought. Earlier transcriptomic studies revealed activation of the HgS2 gene in the leaf of H. glomeratus seedlings when exposed to saline conditions. To identify the properties of HgS2 in H. glomeratus, we used yeast transformation and overexpression in Arabidopsis. Yeast cells genetically transformed with HgS2 exhibited K+ uptake and Na+ efflux compared with control (empty vector). Stable overexpression of HgS2 in Arabidopsis improved its resistance to salt stress and led to a notable rise in seed germination in salinity conditions compared to the wild type (WT). Transgenic Arabidopsis regulated ion homeostasis in plant cells by increasing Na+ absorption and decreasing K+ efflux in leaves, while reducing Na+ absorption and K+ efflux in roots. In addition, overexpression of HgS2 altered transcription levels of stress response genes and regulated different metabolic pathways in roots and leaves of Arabidopsis. These results offer new insights into the role of HgS2 in plants' salt tolerance.

Zero-valent iron (nZVI) nanoparticles mediate SlERF1 expression to enhance cadmium stress tolerance in tomato

Cadmium (Cd) pollution threatens plant physiological and biochemical activities and crop production. Significant progress has been made in characterizing how nanoparticles affect Cd stress tolerance; however, the molecular mechanism of nZVI nanoparticles in Cd stress remains largely uncharacterized. Plants treated with nZVI and exposed to Cd had increased antioxidant capacity and reduced Cd accumulation in plant tissues. The nZVI treatment differentially affected the expression of genes involved in plant environmental responses, including those associated with the ERF transcription factor. SlEFR1 was upregulated by Cd stress in nZVI-treated plants when compared with the control and the predicted protein-protein interactions suggested SlERF1 interacts with proteins associated with plant hormone signaling pathway and related to stress. Yeast overexpressing SlEFR1 grew faster after Cd exposure and significantly had higher Cd stress tolerance when compared with empty vector controls. These results suggest that nZVI induces Cd stress tolerance by activating SlERF1 expression to improve plant growth and nutrient accumulation. Our study reveals the molecular mechanism of Cd stress tolerance for improved plant growth and will support new research on overcoming Cd stress and improving vegetable crop production.

Screening of Saccharomyces cerevisiae metabolite transporters by 13C isotope substrate labeling.

The transportome of Saccharomyces cerevisiae comprises approximately 340 membrane-bound proteins, of which very few are well-characterized. Elucidating transporter proteins' function is essential not only for understanding central cellular processes in metabolite exchange with the external milieu but also for optimizing the production of value-added compounds in microbial cell factories. Here, we describe the application of 13C-labeled stable isotopes and detection by targeted LC-MS/MS as a screening tool for identifying Saccharomyces cerevisiae metabolite transporters. We compare the transport assay's sensitivity, reproducibility, and accuracy in yeast transporter mutant cell lines and Xenopus oocytes. As proof of principle, we analyzed the transport profiles of five yeast amino acid transporters. We first cultured yeast transporter deletion or overexpression mutants on uniformly labeled 13C-glucose and then screened their ability to facilitate the uptake or export of an unlabeled pool of amino acids. Individual transporters were further studied by heterologous expression in Xenopus oocytes, followed by an uptake assay with 13C labeled yeast extract. Uptake assays in Xenopus oocytes showed higher reproducibility and accuracy. Although having lower accuracy, the results from S. cerevisiae indicated the system's potential for initial high-throughput screening for native metabolite transporters. We partially confirmed previously reported substrates for all five amino acid transporters. In addition, we propose broader substrate specificity for two of the transporter proteins. The method presented here demonstrates the application of a comprehensive screening platform for the knowledge expansion of the transporter-substrate relationship for native metabolites in S. cerevisiae.

Up Front Unfolded Protein Response Combined with Early Protein Secretion Pathway Engineering in Yarrowia lipolytica to Attenuate ER Stress Caused by Enzyme Overproduction.

Engineering the yeast Yarrowia lipolytica as an efficient host to produce recombinant proteins remains a longstanding goal for applied biocatalysis. During the protein overproduction, the accumulation of unfolded and misfolded proteins causes ER stress and cell dysfunction in Y. lipolytica. In this study, we evaluated the effects of several potential ER chaperones and translocation components on relieving ER stress by debottlenecking the protein synthetic machinery during the production of the endogenous lipase 2 and the E. coli β-galactosidase. Our results showed that improving the activities of the non-dominant translocation pathway (SRP-independent) boosted the production of the two proteins. While the impact of ER chaperones is protein dependent, the nucleotide exchange factor Sls1p for protein folding catalyst Kar2p is recognized as a common contributor enhancing the secretion of the two enzymes. With the identified protein translocation components and ER chaperones, we then exemplified how these components can act synergistically with Hac1p to enhance recombinant protein production and relieve the ER stress on cell growth. Specifically, the yeast overexpressing Sls1p and cytosolic heat shock protein Ssa8p and Ssb1p yielded a two-fold increase in Lip2p secretion compared with the control, while co-overexpressing Ssa6p, Ssb1p, Sls1p and Hac1p resulted in a 90% increase in extracellular β-galp activity. More importantly, the cells sustained a maximum specific growth rate (μmax) of 0.38 h-1 and a biomass yield of 0.95 g-DCW/g-glucose, only slightly lower than that was obtained by the wild type strain. This work demonstrated engineering ER chaperones and translocation as useful strategies to facilitate the development of Y. lipolytica as an efficient protein-manufacturing platform.

Efficiency of phospholipid remodeling via acyl‐CoA:lysophospholipid acyltransferases action is modified by acyl‐CoAs

AbstractThis study investigated the impact of different acyl‐CoAs on remodeling efficiency of PC and PE via the action of acyl‐CoA:lysophospholipid acyltransferases (LPLAT). Microsomal fractions from yeast ΔALE1 mutant transformed with an empty plasmid pYES2 or plasmid carrying AtLPCAT or ALE1 encoding genes were used in the experiments. In the preliminary assays, presence of 18:1‐CoA in the reaction mixture increased remodeling efficiency of [14C]PC integrated with microsomal fraction of yeast overexpressing AtLPCAT compared to assays without exogenous acyl‐CoA. In further experiments, the effect of five different acyl‐CoAs (16:0‐CoA, 18:0‐CoA, 18:1‐CoA, 18:2‐CoA, and 18:3‐CoA) on remodeling efficiency of yeast microsomal PC and PE via the action of three LPLATs (yeast SLC1 and ALE1 and Arabidopsis LPCAT2) were tested. It has been shown that acyl‐CoAs used in the experiments had different effect on the remodeling intensity of PC and PE via action of the tested acyltransferases. Moreover, each acyl‐CoA used had a different effect on the tested acyltransferases. Additionally, the assays with DTNB (inhibitor of the LPLAT backward reaction) showed that remodeling of PC and PE by LPLATs can also occur through reactions other than the backward reaction carried out by these enzymes, and that acyl‐CoA present in the reaction mixture affected these processes.

BcAMT1;5 Mediates Nitrogen Uptake and Assimilation in Flowering Chinese Cabbage and Improves Plant Growth When Overexpressed in Arabidopsis

Nitrogen (N) is a major limiting factor for plant growth and vegetable production. Understanding the regulatory mechanisms of N uptake, transport, and assimilation is key to improving N use efficiency in plants. Ammonium transporters (AMTs) play an important role in plant N metabolism. In this study, we isolated an important AMT1 subfamily member (BcAMT1;5) with a highly conserved signatural AMT1 subfamily motif from flowering Chinese cabbage. Based on functional complementation in yeast mutant 31019b and overexpression of BcAMT1;5 in Arabidopsis, BcAMT1;5 is a functional AMT. Tissue expression analysis showed that BcAMT1;5 was mainly expressed in roots and showed multiple N regime transcript patterns to respond to varying nutritional conditions. This was up-regulated by N-deficiency and down-regulated by supplying NH4+. The glucuronidase (GUS) activities of BcAMT1;5pro::GUS showed a similar change in response to different N conditions. Overexpression of BcAMT1;5 accelerated the growth of transgenic seedlings, increased NH4+ net influxes, and enhanced the content and accumulation of NH4+ and NO3− at low N concentrations. Additionally, it increased the transcript levels of N assimilation-related genes in shoots. These results indicate that BcAMT1;5 may participate in N uptake and assimilation under various N conditions in flowering Chinese cabbage, but it was differed obviously from other AMT1s.

Genome-Wide Characterization and Function Analysis of ZmERD15 Genes’ Response to Saline Stress in Zea mays L.

Early responsive dehydration (ERD) genes can be rapidly induced by dehydration. ERD15 genes have been confirmed to regulate various stress responses in plants. However, the maize ERD15 members have not been characterized. In the present study, a total of five ZmERD15 genes were identified from the maize genome and named ZmERD15a, ZmERD15b, ZmERD15c, ZmERD15d, and ZmERD15e. Subsequently, their protein properties, gene structure and duplication, chromosomal location, cis-acting elements, subcellular localization, expression pattern, and over-expression in yeast were analyzed. The results showed that the ZmERD15 proteins were characterized by a similar size (113-159 aa) and contained a common domain structure, with PAM2 and adjacent PAE1 motifs followed by an acidic region. The ZmERD15 proteins exhibited a close phylogenetic relationship with OsERD15s from rice. Five ZmERD15 genes were distributed on maize chromosomes 2, 6, 7, and 9 and showed a different exon-intron organization and were expanded by duplication. Besides, the promoter region of the ZmERD15s contained abundant cis-acting elements that are known to be responsive to stress and hormones. Subcellular localization showed that ZmERD15b and ZmERD15c were localized in the nucleus. ZmERD15a and ZmERD15e were localized in the nucleus and cytoplasm. ZmERD15d was localized in the nucleus and cell membrane. The results of the quantitative real-time PCR (qRT-PCR) showed that the expression of the ZmERD15 genes was regulated by PEG, salinity, and ABA. The heterologous expression of ZmERD15a, ZmERD15b, ZmERD15c, and ZmERD15d significantly enhanced salt tolerance in yeast. In summary, a comprehensive analysis of ZmERD15s was conducted in the study. The results will provide insights into further dissecting the biological function and molecular mechanism of ZmERD15s regulating of the stress response in maize.

Overexpression of SmMYC2 enhances salt resistance in Arabidopsis thaliana and Salvia miltiorrhiza hairy roots

Soil salinity significantly affects both Salvia miltiorrhiza growth and development as well as seed germination throughout field cultivation and production. The basic helix-loop-helix (bHLH) transcription factor (TF) MYC2 contributes significantly to plant stress resistance as a key regulator of the jasmonic acid signaling pathway. In transgenic S. miltiorrhiza hairy roots, SmMYC2 has been shown to promote the accumulation of tanshinone and salvianolic acid, but its role in S. miltiorrhiza of resistance to abiotic stress is unclear. Herein, we found methyl jasmonate (MeJA), NaCl, and PEG treatment all significantly increased SmMYC2 expression. In response to salt stress, SmMYC2 overexpression in yeast increased its rate of growth. Additionally, overexpression of SmMYC2 transgenic Arabidopsis thaliana and S. miltiorrhiza hairy root showed that it might improve salt resistance in transgenic plant. In particular, compared to WT, overexpression of SmMYC2 transgenic Arabidopsis had higher levels of three antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)), proline (Pro) content, and ABA-dependent and ABA-independent genes expression. They also had lower levels of malondialdehyde (MDA) and reactive oxygen species (ROS) accumulation. What's more, overexpression of SmMYC2 increases the expression of flavonoid synthesis genes and the accumulation of related components in Arabidopsis. These findings imply that SmMYC2 functions as a positive regulator that regulates plant tolerance to salt through ABA-dependent and independent signaling pathways.

Genome-wide identification of wheat ABC1K gene family and functional dissection of TaABC1K3 and TaABC1K6 involved in drought tolerance.

Activity of BC1 complex kinase (ABC1K) serves as an atypical kinase family involved in plant stress resistance. This study identified 44 ABC1K genes in the wheat genome, which contained three clades (I–III). TaABC1K genes generally had similar structural features, but differences were present in motif and exon compositions from different clade members. More type II functional divergence sites were detected between clade I and clade III and no positive selection site were found in TaABC1K family. The three-dimensional structure prediction by Alphafold2 showed that TaABC1K proteins had more α-helixes with a relatively even distribution, and different clade members had differences in the content of secondary structures. The cis-acting element analysis showed that TaABC1K genes contained abundant cis-acting elements related to plant hormones and environmental stress response in the promoter region, and generally displayed a significantly upregulated expression under drought stress. In particular, both TaABC1K3 and TaABC1K6 genes from clade I was highly induced by drought stress, and their overexpression in yeast and Arabidopsis enhanced drought tolerance by suppressing active oxygen burst and reducing photosynthesis impairment. Meanwhile, TaABC1K3 and TaABC1K6 could, respectively, complement the function of Arabidopsis abc1k3 and abc1k6 mutants and reduce photosynthesis damage caused by drought stress.

Integrated Analysis of Single-Molecule Real-Time Sequencing and Next-Generation Sequencing Eveals Insights into Drought Tolerance Mechanism of Lolium multiflorum.

Lolium multiflorum is widely planted in temperate and subtropical regions globally, and it has high economic value owing to its use as forage grass for a wide variety of livestock and poultry. However, drought seriously restricts its yield and quality. At present, owing to the lack of available genomic resources, many types of basic research cannot be conducted, which severely limits the in-depth functional analysis of genes in L. multiflorum. Therefore, we used single-molecule real-time (SMRT) and next-generation sequencing (NGS) to sequence the complex transcriptome of L. multiflorum under drought. We identified 41,141 DEGs in leaves, 35,559 DEGs in roots, respectively. Moreover, we identified 1243 alternative splicing events under drought. LmPIP5K9 produced two different transcripts with opposite expression patterns, possibly through the phospholipid signaling pathway or the negatively regulated sugar-mediated root growth response to drought stress, respectively. Additionally, 13,079 transcription factors in 90 families were obtained. An in-depth analysis of R2R3-MYB gene family members was performed to preliminarily demonstrate their functions by utilizing subcellular localization and overexpression in yeast. Our data make a significant contribution to the genetics of L. multiflorum, offering a current understanding of plant adaptation to drought stress.

Functional verification and screening of protein interacting with the slPHB3

ABSTRACT slPHB3 was cloned from Salix linearistipularis, the amino acid sequence blast and phylogenetic tree analysis showed that slPHB3 has the most similarity with PHB3 from Populus trichocarpa using DNAMAN software and MEGA7 software. RT-qPCR results confirmed that the expression of slPHB3 was induced obviously under stress treatments. The growth of recombinant yeast cells was better than that of the control group under the stress treatment, indicating that slPHB3 may be involved in the stress response of yeast cells. The transgenic tobacco was treated with different concentrations of NaCl, NaHCO3 and H2O2, fresh weigh of overexpression tobacco were heavier than wild-types. The results showed that transgenic tobacco was more tolerant to salt and oxidation than wild-type tobacco. Expression of important genes including NHX1 and SOS1 in salt stress response pathways are steadily higher in overexpression tobacco than that in wild-types. We identified 17 proteins interacting with slPHB3 by yeast two-hybrid technique, most of these proteins were relation to the stresses. The salt tolerance of slPHB3 expressing yeast and slPHB3 overexpressing plants were better than that of the control. Ten stress-related proteins may interact with slPHB3, which preliminarily indicated that slPHB3 had a certain response relationship with salt stress. The study of slPHB3 under abiotic stress can improve our understanding of PHB3 gene function.

Trichostatin A Relieves Growth Suppression and Restores Histone Acetylation at Specific Sites in a FUS ALS/FTD Yeast Model.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that often occurs concurrently with frontotemporal dementia (FTD), another disorder involving progressive neuronal loss. ALS and FTD form a neurodegenerative continuum and share pathological and genetic features. Mutations in a multitude of genes have been linked to ALS/FTD, including FUS. The FUS protein aggregates and forms inclusions within affected neurons. However, the precise mechanisms connecting protein aggregation to neurotoxicity remain under intense investigation. Recent evidence points to the contribution of epigenetics to ALS/FTD. A main epigenetic mechanism involves the post-translational modification (PTM) of histone proteins. We have previously characterized the histone PTM landscape in a FUS ALS/FTD yeast model, finding a decreased level of acetylation on lysine residues 14 and 56 of histone H3. Here, we describe the first report of amelioration of disease phenotypes by controlling histone acetylation on specific modification sites. We show that inhibiting histone deacetylases, via treatment with trichostatin A, suppresses the toxicity associated with FUS overexpression in yeast by preserving the levels of H3K56ac and H3K14ac without affecting the expression or aggregation of FUS. Our data raise the novel hypothesis that the toxic effect of protein aggregation in neurodegeneration is related to its association with altered histone marks. Altogether, we demonstrate the ability to counter the repercussions of protein aggregation on cell survival by preventing specific histone modification changes. Our findings launch a novel mechanistic framework that will enable alternative therapeutic approaches for ALS/FTD and other neurodegenerative diseases.

Roles of Canavalia rosea metallothioneins in metal tolerance and extreme environmental adaptation to tropical coral reefs

Canavalia rosea (Sw.) DC is a perennial twining herb distributed in the semi-arid and saline–alkali areas of coastal regions and has evolved halotolerance. In this study, we present the first comprehensive survey of the metallothionein (MT) gene family in C. rosea. MT proteins belong to a family of low-molecular-weight polypeptides with a high content of cysteine residues, which have an affinity to bind with heavy metal ions. MTs also play important roles in stress responses as reactive oxygen species (ROS) scavengers. A total of six CrMTs were identified in the C. rosea genome and classified into four subgroups by phylogenetic analysis. An analysis of the cis-acting elements revealed that a series of hormone-, stress-, and development-related cis-acting elements were present in the promoter regions of CrMTs. The expression of CrMTs also showed habitat- and environmental stress-regulated patterns in C. rosea. CrMT overexpression in yeast enhanced tolerance to heavy metals and ROS, as well as high osmotic and alkalinity stress, which is consistent with their predicted roles as metal-chelating proteins and ROS scavengers. Our results indicate that the CrMT genes might contribute to the detoxification of plants to metals and provide marked tolerance against abiotic stress. The expression patterns of CrMTs in C. rosea also indicate that CrMTs play important roles in this species’ response to extreme environments on tropical islands and reefs, probably by improving the thermotolerance of C. rosea plants.

Virulence Vs. Immunomodulation: Roles of the Paracoccin Chitinase and Carbohydrate-Binding Sites in Paracoccidioides brasiliensis Infection.

Paracoccin (PCN) is a bifunctional protein primarily present in the cell wall of Paracoccidioides brasiliensis, a human pathogenic dimorphic fungus. PCN has one chitinase region and four potential lectin sites and acts as both a fungal virulence factor and an immunomodulator of the host response. The PCN activity on fungal virulence, mediated by the chitinase site, was discovered by infecting mice with yeast overexpressing PCN (PCN-ov). PCN-ov are characterized by increased chitin hydrolysis, a narrow cell wall, and augmented resistance to phagocytes' fungicidal activity. Compared to wild-type (wt) yeast, infection with PCN-ov yeast causes a more severe disease, which is attributed to the increased PCN chitinase activity. In turn, immunomodulation of the host response was demonstrated by injecting, subcutaneously, recombinant PCN in mice infected with wt-P. brasiliensis. Through its carbohydrate binding site, the injected recombinant PCN interacts with Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) N-glycans on macrophages, triggers M1 polarization, and stimulates protective Th1 immunity against the fungus. The PCN-treatment of wt yeast-infected mice results in mild paracoccidioidomycosis. Therefore, PCN paradoxically influences the course of murine paracoccidioidomycosis. The disease is severe when caused by yeast that overexpress endogenous PCN, which exerts a robust local chitinase activity, followed by architectural changes of the cell wall and release of low size chito-oligomers. However, the disease is mild when exogenous PCN is injected, which recognizes N-glycans on systemic macrophages resulting in immunomodulation.

Transcriptome and anatomical comparisons reveal the specific characteristics and genes involved in distinct types of growing culms

Moso bamboo can adopt both asexual and sexual reproduction strategies, which result in four main types of growing culms: bamboo shoots, seedling stems, leptomorph rhizomes and outward rhizomes. The genetic mechanisms underlying each type of growing culm remain elusive. Here, we performed comprehensive comparison of the four growing culms at the transcriptional and anatomical levels. Comparative transcriptome analyses showed that candidate genes in the photosynthesis pathway such as Lhca3 and PsbO were highly enriched in the seedling stems. The environmental response genes were highly expressed in outward rhizomes. Besides, genes related to cell cycle processes were predominantly transcribed in bamboo shoots, followed by rhizomes. The anatomical analyses indicated that cell division and elongation in rhizome internodes were only observed in the top section for a short period and barely in the middle and base of rhizome, which differentiate rhizome from other types of culms. In addition, observation on the phenotypic characteristics of winter bamboo shoots also indicated that winter period was important for primary thickening growth, energy storage, and node growth of bamboo shoots. Genes responsible for hormone signalling, cell cycle process, ARF and GRF transcription factors played important roles in this process. In situ hybridization, yeast two-hybrid screening and overexpression in rice indicated that two GRFs interacted with GIFs and played essential roles in moso bamboo shoot growth. In summary, the integration of comprehensive transcriptome profiling and anatomical analyses in this study have shed light on the molecular processes underlying different bamboo culms and provided valuable resources for future genetic research in moso bamboo.

Vacuolar membrane H+-ATPase cˋˋ subunit gene (ThVHAcˋˋ1) from Tamarix hispida Willd improves salt stress tolerance

The plant vacuolar H+-ATPase (V-ATPase) is a multisubunit complex. In addition to performing basic housekeeping functions, this complex is also involved in abiotic stress resistance in plants. In this study, a V-ATPase cˋˋ subunit gene (ThVHAcˋˋ1) from Tamarix hispida Willd was cloned with a 534-bp ORF. Sequence analysis showed that the ThVHAcˋˋ1 protein contains four transmembrane helices and lacks a signal peptide. qRT-PCR results showed that ThVHAcˋˋ1 was primarily induced by treatments of NaCl, NaHCO3, PEG6000, CdCl2 or ABA in roots, stems and leaves of T. hispida. The expression pattern of ThVHAcˋˋ1 was significantly different from that of ThVHAc1 (a V-ATPase c subunit in T. hispida). Furthermore, the cell survival rates and density (OD600) results showed that the transgenic yeast overexpressing ThVHAcˋˋ1 exhibited increased tolerance to the above-mentioned abiotic stresses. In addition, the overexpression of ThVHAcˋˋ1 confers salt tolerance to transgenic Arabidopsis plants by improving the ROS content and decreasing the accumulation of O2− and H2O2. Similarly, the homologous transformation of the ThVHAcˋˋ1 gene into T. hispida also improved salt tolerance. Our results suggest that the ThVHAcˋˋ1 gene plays an important role in plant stress tolerance.

Using Yeast to Identify Coronavirus-Host Protein Interactions.

We have developed a screening system using the yeast Saccharomyces cerevisiae to identify eukaryotic genes involved in the replication of mammalian viruses. Yeast come with various advantages, but in the context of coronavirus research and the system outlined here, they are simple and easy to work with and can be used at biosafety level 2. The system involves inducible expression of individual viral proteins and identification of detrimental phenotypes in the yeast. Yeast knockout and overexpression libraries can then be used for genome-wide screening of host proteins that provide a suppressor phenotype. From the yeast hits, a narrowed list of candidate genes can be produced to investigate for roles in viral replication. Since the system only requires expression of viral proteins, it can be used for any current or emerging virus, regardless of biocontainment requirements and ability to culture the virus. In this chapter, we will outline the protocols that can be used to take advantage of S. cerevisiae as a tool to advance understanding of how viruses interact with eukaryotic cells.