A Redox-Sensitive Cysteine Is Required for PIN1At Function

Parvulins are ubiquitous peptidyl-prolyl isomerases (PPIases) required for protein folding and regulation. Among parvulin members, Arabidopsis PIN1At, human PIN1, and yeast ESS1 share a conserved cysteine residue but differ by the presence of an N-terminal WW domain, absent in PIN1At. In this study, we have explored whether the cysteine residue of Arabidopsis PIN1At is involved in catalysis and subject to oxidative modifications. From the functional complementation of yeast ess1 mutant, we concluded that the cysteine at position 69 is mandatory for PIN1At function in vivo, unless being replaced by an Asp which is found in a few parvulin members. This result correlates with a decrease of the in vitro PPIase activity of non-functional PIN1At cysteinic variants. A decrease of PIN1At activity was observed upon H2O2 treatment. The in vitro oxidation of cysteine 69, which has an acidic pKa value of 4.9, leads to the formation of covalent dimers that are reduced by thioredoxins, or to sulfinic or sulfonic acid forms at higher H2O2 excess. These investigations highlight the importance of the sole cysteine residue of PIN1At for activity. The reversible formation of an intermolecular disulfide bond might constitute a protective or regulatory mechanism under oxidizing conditions.

Functional Gene Identification and Corresponding Tolerant Mechanism of High Furfural-Tolerant Zymomonas mobilis Strain F211

Furfural is a major inhibitor in lignocellulose hydrolysate for Zymomonas mobilis. A mutant F211 strain with high furfural tolerance was obtained from our previous study. Thus, its key tolerance mechanism was studied in the present study. The function of mutated genes in F211 was identified by functional complementation experiments, revealing that the improved furfural tolerance was resulted from the C493T mutation of the ZCP4_0270 gene promoting cell flocculation and the mutation (G1075A)/downregulation of ZCP4_0970. Comparative transcriptome analysis revealed 139 differentially expressed genes between F211 and the control, CP4, in response to furfural stress. In addition, the reliability of the RNA-Seq data was also confirmed. The potential tolerance mechanism was further demonstrated by functional identification of tolerance genes as follows: (I) some upregulated or downregulated genes increase the levels of NAD(P)H, which is involved in the reduction of furfural to less toxic furfuryl alcohol, thus accelerating the detoxification of furfural; (II) the mutated ZCP4_0270 and upregulated cellulose synthetase gene (ZCP4_0241 and ZCP4_0242) increased flocculation to resist furfural stress; (III) upregulated molecular chaperone genes promote protein synthesis and repair stress-damaged proteins; and (IV) transporter genes ZCP4_1623–1,625 and ZCP4_1702–1703 were downregulated, saving energy for cell growth. The furfural-tolerant mechanism and corresponding functional genes were revealed, which provides a theoretical basis for developing robust chassis strains for synthetic biology efforts.

Cloning and expression analysis of mevalonate kinase and phosphomevalonate kinase genes associated with the MVA pathway in Santalum album

Sandalwood (Santalum album L.) is highly valued for its fragrant heartwood and extracted oil. Santalols, which are the main components of that oil, are terpenoids, and these are biosynthesized via the mevalonic acid (MVA) pathway. Mevalonate kinase (MK) and phosphomevalonate kinase (PMK) are key enzymes in the MVA pathway. Little is known about the genes that encode MK and PMK in S. album or the mechanism that regulates their expression. To isolate and identify the functional genes involved in santalol biosynthesis in S. album, an MK gene designated as SaMK, and a PMK gene designated as SaPMK, were cloned from S. album. The sequences of these genes were analyzed. A bioinformatics analysis was conducted to assess the homology of SaMK and SaPMK with MK and PMK genes from other plants. The subcellular localization of SaMK and SaPMK proteins was also investigated, as was the functional complementation of SaMK and SaPMK in yeast. Our results show that the full-length cDNA sequences of SaMK and SaPMK were 1409 bp and 1679 bp long, respectively. SaMK contained a 1381 bp open reading frame (ORF) encoding a polypeptide of 460 amino acids and SaPMK contained a 1527 bp ORF encoding a polypeptide of 508 amino acids. SaMK and SaPMK showed high homology with MK and PMK genes of other plant species. Functional complementation of SaMK in a MK-deficient mutant yeast strain YMR208W and SaPMK in a PMK-deficient mutant yeast strain YMR220W confirmed that cloned SaMK and SaPMK cDNA encode a functional MK and PMK, respectively, mediating MVA biosynthesis in yeast. An analysis of tissue expression patterns revealed that SaMK and SaPMK were constitutively expressed in all the tested tissues. SaMK was highly expressed in young leaves but weakly expressed in sapwood. SaPMK was highly expressed in roots and mature leaves, but weakly expressed in young leaves. Induction experiments with several elicitors showed that SaMK and SaPMK expression was upregulated by methyl jasmonate. These results will help to further study the role of MK and PMK genes during santalol biosynthesis in S. album.

Horizontal transfer of a conserved npc-2 like effector gene in rust fungi that suppresses cell death in plants

ML/MD-2 is a conserved lipid/sterol-binding protein family having a role in sterol transfer and innate immunity in lower and higher eukaryotes. Here we report a genome-wide survey of this family, identifying 84 genes in 25 fungal and five oomycetes plant pathogen, having a different nutrition mode. All the fungal species were found to have varied numbers of family members, a distinctively substantial expansion of the ML gene family was observed in Rhizophagus irregularis (RI) with 33 genes. Our analysis also showed that NPC2 like proteins, a subfamily of ML domain superfamily, were not only restricted to animals and insect species but also present in plant fungal pathogens, including members of Clavicipitaceae, Pucciniacease, and Tremellaceae family. The phylogenetic analysis showed that these NPC2 like fungal proteins are more closely related to animals/insects than other fungal species. The molecular docking studies of these proteins with cholesterol and other derivatives indicate lipid-binding functional conservation across the animal and fungi kingdom. Further, the full length CDS of one of the npc2 like genes from Puccinia triticina (Pt5643) was PCR amplified and further characterized using various studies such as qRT-PCR, expression in onion epidermal cells, Nicotiana benthamiana for subcellular localization studies, yeast functional complementation, and expression studies. The mRNA abundance of Pt5643 was observed to be increased along with the infection progression and exhibits the highest expression at 5thday post-infection (dpi), suggesting its important role in the P. triticina infection cycle in wheat. The fluorescent confocal microscopy of transiently expressed YFP tagged Pt5643 in onion epidermal cells and N. benthamiana shows its location in cytoplasm and nucleus, indicating its involvement in the manipulation of host genes. The functional complementation of Pt5643 in npc2 mutant yeast showed its functional similarity to the eukaryotic npc2 gene. Further, the overexpression of Pt5643 also suppressed the BAX and H2O2 induced program cell death in N. benthamiana and yeast, respectively thus proving to be a novel horizontally transferred effector in rust fungal pathogens. Altogether the present study reports the novel function of fungal NPC2 like proteins playing a crucial role in host defense manipulation possibly through lipid binding/transport similar to animals.

FANCD2-associated nuclease 1 partially compensates for the lack of Exonuclease 1 in mismatch repair.

Germline mutations in the mismatch repair ( MM R) genes MSH2 , MSH6 , MLH1 and PMS2 are linked to cancer of the colon and other organs, characterised by microsatellite instability and a large increase in mutation frequency. Unexpectedly, mutations in EXO1 , encoding the only exonuclease genetically implicated in MMR, are not linked to familial cancer and cause a substantially weaker mutator phenotype. This difference could be explained if eukaryotic cells possessed additional exonucleases redundant with EXO1. Analysis of the MLH1 interactome identified FANCD2-associated nuclease 1 (FAN1), a novel enzyme with biochemical properties resembling EXO1. We now show that FAN1 efficiently substitutes for EXO1 in MMR assays and that this functional complementation is modulated by its interaction with MLH1. FAN1 also contributes towards MMR in vivo : cells lacking both EXO1 and FAN1 have a MMR defect and display resistance to N -methyl- N -nitrosourea (MNU) and 6-thioguanine (TG). Moreover, FAN1 loss amplifies the mutational profile of EXO1-deficient cells, implying that the two nucleases act redundantly in the same antimutagenic pathway. However, the increased drug resistance and mutator phenotype of FAN1/EXO1-deficient cells are less prominent than those seen in cells lacking MSH6 or MLH1. Eukaryotic cells thus apparently possess additional mechanisms that compensate for the loss of EXO1.

The Sugar Porter gene family of Piriformospora indica: Nomenclature, Transcript Profiling and Characterization

Piriformospora indica is one of the prominent mutualistic root endophyte known to overcome phosphate and nitrogen limitation in a wide variety of plant species, reciprocally takes up carbohydrates for its survival and growth. A total of nineteen potential hexose transporters have been identified from P. indica genome, that may contributes to its potential of carbohydrate assimilation from host plant. Phylogenetic analysis assembles it in 10 groups within 3 clusters. To ease the study, systematic nomenclature were provided to 19 putative hexose transporters as “PiST1-PiST19” in accordance to their appearance on the supercontigs genome sequence of P. indica. The protein length ranges from 487 to 608 amino acids. Out of 19 putative hexose transporters, 9 have been predicted to contain 12 transmembrane domains (PiST1, PiST2, PiST5, PiST6, PiST9, PiST10, PiST11, PiST12 and PiST17), along with MFS family and Sugar porter subfamily motif. Therefore, transcripts were detected for these 9 genes. During colonization, three P. indica genes PiST1, PiST5 and PiST9 have shown induction as compared to axenic culture. Similarly during phosphate starvation, revealed PiST12 to be strongly enhanced. Carbon starvation study in liquid axenic culture resulted in induction of 4 genes, PiST6, PiST9, PiST12 and PiST17. We found co-relation in the expression pattern of PiPT and PiST12 during phosphate starvation. In silico analysis revealed the presence of functional conserved fucose permease (FucP) domain, involved in fructose transport. Phylogenetic analysis revealed that PiST12 groups closely with basidiomycetes hexose transporters. Further, functional complementation of Δhxt null mutant revealed, PiST12 is able to complement growth on fructose and galactose but negligible on glucose.

Characterization of type-2 diacylglycerol acyltransferases in Haematococcus lacustris reveals their functions and engineering potential in triacylglycerol biosynthesis

Background Haematococcus lacustris is an ideal source of astaxanthin (AST), which is stored in oil bodies containing esterified AST (EAST) and triacylglycerol (TAG). Diacylglycerol acyltransferases (DGATs) catalyze the last step of acyl-CoA-dependent TAG biosynthesis and are also considered as crucial enzymes involved in EAST biosynthesis in H. lacustris. Previous studies have identified four putative DGAT2-encoding genes in H. lacustris, and only HpDGAT2D allowed the recovery of TAG biosynthesis, but the engineering potential of HpDGAT2s in TAG biosynthesis remains ambiguous. Results Five putative DGAT2 genes (HpDGAT2A, HpDGAT2B, HpDGAT2C, HpDGAT2D, and HpDGAT2E) were identified in H. lacustris. Transcription analysis showed that the expression levels of the HpDGAT2A, HpDGAT2D, and HpDGAT2E genes markedly increased under high light and nitrogen deficient conditions with distinct patterns, which led to significant TAG and EAST accumulation. Functional complementation demonstrated that HpDGAT2A, HpDGAT2B, HpDGAT2D, and HpDGAT2E had the capacity to restore TAG synthesis in a TAG-deficient yeast strain (H1246) showing a large difference in enzymatic activity. Fatty acid (FA) profile assays revealed that HpDGAT2A, HpDGAT2D, and HpDGAT2E, but not HpDGAT2B, preferred monounsaturated fatty acyl-CoAs (MUFAs) for TAG synthesis in yeast cells, and showed a preference for polyunsaturated fatty acyl-CoAs (PUFAs) based on their feeding strategy. The heterologous expression of HpDGAT2D in Arabidopsis thaliana and Chlamydomonas reinhardtii significantly increased the TAG content and obviously promoted the MUFAs and PUFAs contents. Conclusions Our study represents systematic work on the characterization of HpDGAT2s by integrating expression patterns, AST/TAG accumulation, functional complementation, and heterologous expression in yeast, plants, and algae. These results (1) update the gene models of HpDGAT2s, (2) prove the TAG biosynthesis capacity of HpDGAT2s, (3) show the strong preference for MUFAs and PUFAs, and (4) offer target genes to modulate TAG biosynthesis by using genetic engineering methods.

Cloning and Expression Analysis of Mevalonate Kinase and Phosphomevalonate Kinase Genes Associated with MVA Pathway in Santalum Album

Sandalwood is highly valued for its fragrant heartwood and its extracted oil. The major oil component santalols are terpenoids, which are biosynthesis through the MVA pathway. MK and PMK are the major enzymes on the MVA pathway. Little is known about the genes encoding MK and PMK in Santalum album on its expression regulation mechanism. The analysis of MK and PMK genes and their functions are important for the further study of the biosynthesis of santalol. These results will help to further study the role of MK and PMK genes in S. album santalol biosynthesis. The total RNA of sandalwood leaves was extracted, then the First-strand cDNA synthesis was obtained through the PrimeScript first-strand cDNA synthesis kit. Then sequence comparison and bioinformatics analyses of the genes homology of SaMK and SaPMK with MKs and PMKs, We also investigated subcellular localization of SaMK and SaPMK proteins. Its functional complementation of SaMK and SaPMK in yeast were also investigated. Atlast, MeJA was used to induce tissue-specific analysis and expression profiles of SaMK and SaPMK. The results showed that the full-length cDNA sequences of SaMK and SaPMK were 1409 bp and 1679 bp containing a 1381 bp open reading frame (ORF) encoding a polypeptide of 460 amino acids and a 1527 bp ORF encoding a polypeptide of 508 amino acids, respectively. Sequence comparison and bioinformatics analyses indicated that SaMK and SaPMK showed high homology with MKs and PMKs, respectively from other plant species. Further functional complementation of SaMK in an MK-deficient mutant yeast strain YMR208W and SaPMK in a PMK-deficient mutant yeast strainYMR220W confirmed that cloned SaMK and SaPMK cDNA encode a functional MK and PMK, respectively and mediated MVA biosynthesis in yeast. Tissue expression pattern analysis revealed that SaMK and SaPMK were constitutively expressed in all the tested tissues. SaMK was highly expressed in young leaves but least expressed in sapwood while SaPMK was highly expressed in roots and mature leaves, and least expressed in young leaves.

Characterization of type-2 diacylglycerol acyltransferases in Haematococcus lacustris reveals their functions and engineering potential in triacylglycerol biosynthesis

Background: Haematococcus lacustris is an ideal source of astaxanthin (AST), which is stored in oil bodies containing esterified AST (EAST) and triacylglycerol (TAG). Diacylglycerol acyltransferases (DGATs) catalyze the last step of acyl-CoA-dependent TAG biosynthesis and are also considered as crucial enzymes involved in EAST biosynthesis in H. lacustris. Previous studies have identified four putative DGAT2-encoding genes in H. lacustris, and only HpDGAT2D allowed the recovery of TAG biosynthesis, but the engineering potential of HpDGAT2s in TAG biosynthesis remains ambiguous.Results: Five putative DGAT2 genes (HpDGAT2A, HpDGAT2B, HpDGAT2C, HpDGAT2D, and HpDGAT2E) were identified in H. lacustris. Transcription analysis showed that the expression levels of the HpDGAT2A, HpDGAT2D, and HpDGAT2E genes markedly increased under high light and nitrogen deficient conditions with distinct patterns, which led to significant TAG and EAST accumulation. Functional complementation demonstrated that HpDGAT2A, HpDGAT2B, HpDGAT2D, and HpDGAT2E had the capacity to restore TAG synthesis in a TAG-deficient yeast strain (H1246) showing a large difference in enzymatic activity. Fatty acid (FA) profile assays revealed that HpDGAT2A, HpDGAT2D, and HpDGAT2E, but not HpDGAT2B, preferred monounsaturated fatty acyl-CoAs (MUFAs) for TAG synthesis in yeast cells, and showed a preference for polyunsaturated fatty acyl-CoAs (PUFAs) based on their feeding strategy. The heterologous expression of HpDGAT2D in Arabidopsis thaliana and Chlamydomonas reinhardtii significantly increased the TAG content and obviously promoted the MUFAs and PUFAs contents.Conclusions: Our study represents systematic work on the characterization of HpDGAT2s by integrating expression patterns, AST/TAG accumulation, functional complementation, and heterologous expression in yeast, plants, and algae. These results (1) update the gene models of HpDGAT2s, (2) prove the TAG biosynthesis capacity of HpDGAT2s, (3) show the strong preference for MUFAs and PUFAs, and (4) offer target genes to modulate TAG biosynthesis by using genetic engineering methods.