Plant-specific environmental and developmental signals regulate the mismatch repair protein MSH6 in Arabidopsis thaliana.

The DNA mismatch repair (MMR) is a postreplicative system that guarantees genomic stability by correcting mispaired and unpaired nucleotides. In eukaryotic nuclei, MMR is initiated by the binding of heterodimeric MutS homologue (MSH) complexes to the DNA error or lesion. Among these proteins, MSH2-MSH6 is the most abundant heterodimer. Even though the MMR mechanism and proteins are highly conserved throughout evolution, physiological differences between species can lead to different regulatory features. Here, we investigated how light, sugar, and/or hormones modulate Arabidopsis thaliana MSH6 expression pattern. We first characterized the promoter region of MSH6. Phylogenetic shadowing revealed three highly conserved regions. These regions were analyzed by the generation of deletion constructs of the MSH6 full-length promoter fused to the ß-glucuronidase (GUS) gene. Combined, our in silico and genetic analyses revealed that a 121-bp promoter fragment was necessary for MSH6 expression and contained potential cis-acting elements involved in light- and hormone-responsive gene expression. Accordingly, light exposure or sugar treatment of four-day old A. thaliana seedlings triggered an upregulation of MSH6 in shoot and root apical meristems. Appropriately, MSH6 was also induced by the stem cell inducer WUSCHEL. Further, the stimulatory effect of light was dependent on the presence of phyA. In addition, treatment of seedlings with auxin or cytokinin also caused an upregulation of MSH6 under darkness. Consistent with auxin signals, MSH6 expression was suppressed in the GATA23 RNAi line compared with the wild type. Our results provide evidence that endogenous factors and environmental signals controlling plant growth and development regulate the MSH6 protein in A. thaliana.

Exploring soybean cultivar susceptibility to sudden death syndrome: Insights into isoflavone responses and biocontrol potential.

Sudden Death Syndrome (SDS) caused by Fusarium tucumaniae is a significant threat to soybean production in Argentina. This study assessed the susceptibility of SY 3 × 7 and SPS 4 × 4 soybeans cultivars to F. tucumaniae and studied changes in root isoflavone levels after infection. Additionally, the biocontrol potential of plant-growth promoting rhizobacteria (PGPR) against SDS was also examined. Our results demonstrated that the SY 3 × 7 cultivar exhibited higher disease severity and total fresh weight loss than SPS 4 × 4. Both cultivars showed induction of daidzein, glycitein, and genistein in response to infection, with the partially resistant cultivar displaying significantly higher daidzein levels than the susceptible cultivar at 14 days post infection (dpi) (2.74 vs 2.17-fold), declining to a lesser extent at 23 dpi (0.94 vs 0.35-fold, respectively). However, daidzein was not able to inhibit F. tucumaniae growth in in vitro assays probably due to its conversion to an isoflavonoid phytoalexin which would ultimately be an effective fungal inhibitor. Furthermore, the PGPR bacterium Bacillus amyloliquefaciens BNM340 displayed antagonistic activity against F. tucumaniae and reduced SDS symptoms in infected plants. This study sheds light on the varying susceptibility of soybean cultivars to SDS, offers insights into isoflavone responses during infection, and demonstrates the potential of PGPR as a biocontrol strategy for SDS management, providing ways for disease control in soybean production.

Deficiency of the Arabidopsis mismatch repair MSH6 attenuates Pseudomonas syringae invasion.

The MutS homolog 6 (MSH6) is a nuclear DNA mismatch repair (MMR) gene that encodes the MSH6 protein. MSH6 interacts with MSH2 to form the MutSα heterodimer. MutSα corrects DNA mismatches and unpaired nucleotides arising during DNA replication, deamination of 5-methylcytosine, and recombination between non-identical DNA sequences. In addition to correcting DNA biosynthetic errors, MutSα also recognizes chemically damaged DNA bases. Here, we show that inactivation of MSH6 affects the basal susceptibility of Arabidopsis thaliana to Pseudomonas syringae pv tomato DC3000. The msh6 T-DNA insertional mutant exhibited a reduced susceptibility to the bacterial invasion. This heightened basal resistance of msh6 mutants appears to be dependent on an increased stomatal closure, an accumulation of H2O2 and double-strand breaks (DSBs) and a constitutive expression of pathogenesis-related (NPR1 and PR1) and DNA damage response (RAD51D and SOG1) genes. Complementation of this mutant with the MSH6 wild type allele under the control of its own promoter resulted in reversal of the basal bacterial resistance phenotype and the stomatal closure back to wild type levels. Taken together, these results demonstrate that inactivation of MSH6 increases Arabidopsis basal susceptibility to the bacterial pathogen and suggests a link between DNA repair and stress signaling in plants.

Role of the mismatch repair protein MSH7 in Arabidopsis adaptation to acute salt stress.

DNA mismatch repair (MMR) is a highly conserved pathway in evolution responsible for maintaining genomic stability. MMR is initiated when MutS proteins recognize and repair single base-base mismatches and small loops of unpaired nucleotides as well as certain types of DNA damage. Arabidopsis thaliana and other plants contain MutS protein homologs (MSH) found in other eukaryotic organisms and a unique MSH7 polypeptide. In this study, we first evaluated transient expression profiles of ten-days old pAtMSH7:GUS transgenic seedlings at different recovery times after an acute treatment for 48 hs with100 mM NaCl. GUS histochemical staining indicated that MSH7 expression is repressed by salt exposure but recovers progressively. Then, ten-days old mutants harboring two independent msh7 alleles were exposed for 48 hs with100 mM NaCl and different traits were measured over recovery time. Salt treated msh7 seedlings were defective in G2/M arrest. As a result, msh7 seedlings showed a reduced salt inhibitory effect as evidenced by a decreased reduction of rosette and leaf areas, stomatal density, total leaf number, silique length and seed number per silique. These findings suggest that disruption of MSH7 activity could be a promising approach for plant adaptive responses to salinity stress.

Ribosomal Protein RPL10A Contributes to Early Plant Development and Abscisic Acid-Dependent Responses in Arabidopsis.

Plant ribosomal proteins play universal roles in translation, although they are also involved in developmental processes and hormone signaling pathways. Among Arabidopsis RPL10 family members, RPL10A exhibits the highest expression during germination and early development, suggesting that RPL10A is the main contributor to these processes. In this work, we first analyzed RPL10A expression pattern in Arabidopsis thaliana using transgenic RPL10Apro:GUS plants. The gene exhibits a ubiquitous expression pattern throughout the plant, but it is most strongly expressed in undifferentiated tissues. Interestingly, gene expression was also detected in stomatal cells. We then examined protein function during seedling establishment and abscisic acid (ABA) response. Heterozygous rpl10A mutant plants show decreased ABA-sensitivity during seed germination, are impaired in early seedling and root development, and exhibit reduced ABA-inhibition of stomatal aperture under light conditions. Overexpression of RPL10A does not affect the germination and seedling growth, but RPL10A-overexpressing lines are more sensitive to ABA during early plant development and exhibit higher stomatal closure under light condition both with and without ABA treatment than wild type plants. Interestingly, RPL10A expression is induced by ABA. Together, we conclude that RPL10A could act as a positive regulator for ABA-dependent responses in Arabidopsis plants.

Optimization of protease production and sequence analysis of the purified enzyme from the cold adapted yeast Rhodotorula mucilaginosa CBMAI 1528.

Enzymes from cold-adapted microorganisms are of high interest to industries due to their high activity at low and mild temperatures, which makes them suitable for their use in several processes that either require a supply of exogenous energy or involve the use of heat labile products. In this work, the protease production by the strain Rhodotorula mucilaginosa CBMAI 1528, previously isolated from the Antarctic continent, was optimized, and the purified enzyme analyzed. It was found that protease production was dependent on culture medium composition and growth temperature, being 20 °C and a culture medium containing both glucose and casein peptone (20 and 10 g/L, respectively) the optimal growing conditions in batch as well as in bioreactor. Moreover, mass spectrometry analysis revealed that the enzyme under study has a 100 % sequence identity with the deduced amino acid sequence of a putative aspartic protease from Rhodotorula sp. JG-1b (protein ID: KWU42276.1). This result was confirmed by the decrease of 95 % proteolytic activity by pepstatin A, a specific inhibitor of aspartic proteases. We propose that the enzyme reported here could be Rodothorulapepsin, a protein characterized in 1972 that did not have an associated sequence to date and has been classified as an orphan enzyme.

The mismatch repair protein MSH6 regulates somatic recombination in Arabidopsis thaliana.

The mismatch repair (MMR) pathway promotes genome stability by controlling the fidelity of replication and recombination. The first step of the pathway involves recognition of the mismatch by heterodimers composed of MutS homologs (MSH). Although MSH6 has been well characterized in yeasts and humans, the role of the plant protein has not been extensively studied. We first analyzed gene expression in Arabidopsis thaliana. The use of transgenic plants expressing the ß-glucuronidase (GUS) reporter gene under the control of approximately 1-kb region upstream of the start codon of the AtMSH6 gene demonstrated that MSH6 is preferentially expressed in undifferentiated cells with an intense cell division rate. We then examined protein function in meiotic and somatic recombination. Suppression of AtMSH6 did not affect the rate of meiotic recombination, but increased the frequency of recombination between two homeologous repeats of a marker gene by 3-fold relative to wild-type plants. Expression of the AtMSH6 gene under the control of its own promoter in msh6 homozygous mutant plants rescued the altered somatic recombination phenotype. We conclude that MSH6 shows a functional conservation across different biological kingdoms and a functional specificity in plants.

Growth and development of AtMSH7 mutants in Arabidopsis thaliana.

DNA mismatch repair (MMR) is a highly conserved biological pathway that improves the fidelity of DNA replication and recombination. MMR is initiated when MutS proteins recognize mismatches and small loops of unpaired nucleotides. Arabidopsis thaliana and other plants encode MutS protein homologs (MSH) conserved among other eukaryotic organisms, but also encode an extra MSH polypeptide (MSH7). In order to better understand the role of MSH7 in vivo, a full set of phenotypic parameters that covered the development of the plant from seed imbibition to flowering and seed maturation was analyzed in A. thaliana harboring two different msh7 alleles. Plants deficient in MSH7 show statistically significant faster germination rates, longer primary roots during the juvenile vegetative phase, and higher cauline leaf and axillary and lateral inflorescence numbers compared with wild type. We also quantified number, length and area of siliques and seed number per silique. Disruption of MSH7 resulted in a higher number of smaller siliques than wild type. There were no differences in seed number per silique between genotypes. These findings suggest that mutant plant growth appears to be caused by an impaired cell cycle checkpoint that allows cell division without adequate DNA repair. This increase in proliferation activity demonstrates a functional and temporal link between DNA repair and cell cycle regulation.

Primary metabolism changes triggered in soybean leaves by Fusarium tucumaniae infection.

Sudden death syndrome (SDS) of soybean can be caused by at least four distinct Fusarium species, with F. tucumaniae being the main causal agent in Argentina. The fungus is a soil-borne pathogen that is largely confined to the roots, but damage also reaches aerial part of the plant and interveinal chlorosis and necrosis, followed by premature defoliation can be observed. In this study, two genetically diverse soybean cultivars, one susceptible (NA 4613) and one partially resistant (DM 4670) to SDS infection, were inoculated with F. tucumaniae or kept uninoculated. Leaf samples at 7, 10, 14 and 25 days post-inoculation (dpi) were chosen for analysis. With the aim of detecting early markers that could potentially discriminate the cultivar response to SDS, gas chromatography-mass spectrometry (GC-MS) analyses and biochemical studies were performed. Metabolic analyses show higher levels of several amino acids in the inoculated than in the uninoculated susceptible cultivar starting at 10 dpi. Biochemical studies indicate that pigment contents and Rubisco level were reduced while class III peroxidase activity was increased in the inoculated susceptible plant at 10 dpi. Taken together, our results indicate that the pathogen induced an accumulation of amino acids, a decrease of the photosynthetic activity, and an increase of plant-specific peroxidase activity in the susceptible cultivar before differences of visible foliar symptoms between genotypes could be observed, thus suggesting that metabolic and biochemical approaches may contribute to a rapid characterization of the cultivar response to SDS.

Protecting DNA from errors and damage: an overview of DNA repair mechanisms in plants compared to mammals.

The genome integrity of all organisms is constantly threatened by replication errors and DNA damage arising from endogenous and exogenous sources. Such base pair anomalies must be accurately repaired to prevent mutagenesis and/or lethality. Thus, it is not surprising that cells have evolved multiple and partially overlapping DNA repair pathways to correct specific types of DNA errors and lesions. Great progress in unraveling these repair mechanisms at the molecular level has been made by several talented researchers, among them Tomas Lindahl, Aziz Sancar, and Paul Modrich, all three Nobel laureates in Chemistry for 2015. Much of this knowledge comes from studies performed in bacteria, yeast, and mammals and has impacted research in plant systems. Two plant features should be mentioned. Plants differ from higher eukaryotes in that they lack a reserve germline and cannot avoid environmental stresses. Therefore, plants have evolved different strategies to sustain genome fidelity through generations and continuous exposure to genotoxic stresses. These strategies include the presence of unique or multiple paralogous genes with partially overlapping DNA repair activities. Yet, in spite (or because) of these differences, plants, especially Arabidopsis thaliana, can be used as a model organism for functional studies. Some advantages of this model system are worth mentioning: short life cycle, availability of both homozygous and heterozygous lines for many genes, plant transformation techniques, tissue culture methods and reporter systems for gene expression and function studies. Here, I provide a current understanding of DNA repair genes in plants, with a special focus on A. thaliana. It is expected that this review will be a valuable resource for future functional studies in the DNA repair field, both in plants and animals.

Role of AtMSH7 in UV-B-induced DNA damage recognition and recombination.

The mismatch repair (MMR) system maintains genome integrity by correcting replication-associated errors and inhibiting recombination between divergent DNA sequences. The basic features of the pathway have been highly conserved throughout evolution, although the nature and number of the proteins involved in this DNA repair system vary among organisms. Plants have an extra mismatch recognition protein, MutSγ, which is a heterodimer: MSH2-MSH7. To further understand the role of MSH7 in vivo, we present data from this protein in Arabidopsis thaliana. First, we generated transgenic plants that express ß-glucuronidase (GUS) under the control of the MSH7 promoter. Histochemical staining of the transgenic plants indicated that MSH7 is preferentially expressed in proliferating tissues. Then, we identified msh7 T-DNA insertion mutants. Plants deficient in MSH7 show increased levels of UV-B-induced cyclobutane pyrimidine dimers relative to wild-type (WT) plants. Consistent with the patterns of MSH7 expression, we next analysed the role of the protein during somatic and meiotic recombination. The frequency of somatic recombination between homologous or homeologous repeats (divergence level of 1.6%) was monitored using a previously described GUS recombination reporter assay. Disruption of MSH7 has no effect on the rates of somatic homologous or homeologous recombination under control conditions or after UV-B exposure. However, the rate of meiotic recombination between two genetically linked seed-specific fluorescent markers was 97% higher in msh7 than in WT plants. Taken together, these results suggest that MSH7 is involved in UV-B-induced DNA damage recognition and in controlling meiotic recombination.

Metabolic profiles of soybean roots during early stages of Fusarium tucumaniae infection.

Soybean germplasm exhibits various levels of resistance to Fusarium tucumaniae, the main causal agent of sudden death syndrome (SDS) of soybean in Argentina. In this study, two soybean genotypes, one susceptible (NA 4613) and one partially resistant (DM 4670) to SDS infection, were inoculated with F. tucumaniae. Disease symptoms were scored at 7, 10, 14, and 25 days post-inoculation (dpi). The greatest difference in the area under the disease progress curve (AUDPC) values among genotypes was observed at 25 dpi. In order to detect early metabolic markers that could potentially discriminate between susceptible and resistant genotypes, gas chromatography-mass spectrometry (GC-MS) analyses of root samples were performed. These analyses show higher levels of several amino acids and the polyamine cadaverine in the inoculated than in the uninoculated susceptible cultivar at 7 dpi. Principal component analysis (PCA) revealed that the metabolic profile of roots harvested at the earliest time points from the inoculated susceptible genotype was clearly differentiated from the rest of the samples. Furthermore, variables associated with the first principal component were mainly amino acids. Taken together, the results indicate that the pathogen induced the susceptible plant to accumulate amino acids in roots at early time points after infection, suggesting that GC-MS-based metabolomics could be used for the rapid characterization of cultivar response to SDS.

Genetic response of Paspalum plicatulum to genome duplication.

Paspalum plicatulum is a perennial rhizomatous grass with natural diploid and polyploid cytotypes. In this study, we investigated the occurrence of sequence polymorphisms arising immediately after genome autoduplication in this species. Two mixoploid plants (4C and 7D) were previously obtained through colchicine treatment of seeds generated by open pollination of a diploid plant (H14-2x). Diploid and tetraploid sectors from both mixoploids were dissected to generate two ploidy series (4C-2x/4C-4x and 7D-2x/7D-4x). Molecular fingerprints were generated from the maternal plant H14-2x, both ploidy series (4C-2x/4C-4x and 7D-2x/7D-4x), and a tetraploid plant (C1) produced by selfing 7D-4x. Our results indicate that immediately after polyploidization P. plicatulum suffers genetic rearrangements affecting ~28-38 % of the genome. Band gain and loss were equally prevalent at a statistically significant level. At least 5.62 % of the genome experimented recurrent genetic variation in a non-random basis with a confidence of 94.88 %. A significant proportion of novel bands (36 out of 195; 18.4 %) was detected in the C1 tetraploid plant. Half of these bands were not amplified in either H14-2x or 7D-4x, while the remainders were present in H14-2x but absent in 7D-4x. Our results indicate the occurrence of a considerable number of genetic changes in P. plicatulum immediately after polyploidization, some of which were recurrently detected in different independent events. Moreover, we confirmed that after polyploidization, lost ancestral alleles were spontaneously recovered in further generations, a phenomenon previously reported by other research groups.

Candida infections, causes, targets, and resistance mechanisms: traditional and alternative antifungal agents.

The genus Candida includes about 200 different species, but only a few species are human opportunistic pathogens and cause infections when the host becomes debilitated or immunocompromised. Candida infections can be superficial or invasive. Superficial infections often affect the skin or mucous membranes and can be treated successfully with topical antifungal drugs. However, invasive fungal infections are often life-threatening, probably due to inefficient diagnostic methods and inappropriate initial antifungal therapies. Here, we briefly review our current knowledge of pathogenic species of the genus Candida and yeast infection causes and then focus on current antifungal drugs and resistance mechanisms. An overview of new therapeutic alternatives for the treatment of Candida infections is also provided.

Molecular fingerprints to identify Candida species.

A wide range of molecular techniques have been developed for genotyping Candida species. Among them, multilocus sequence typing (MLST) and microsatellite length polymorphisms (MLP) analysis have recently emerged. MLST relies on DNA sequences of internal regions of various independent housekeeping genes, while MLP identifies microsatellite instability. Both methods generate unambiguous and highly reproducible data. Here, we review the results achieved by using these two techniques and also provide a brief overview of a new method based on high-resolution DNA melting (HRM). This method identifies sequence differences by subtle deviations in sample melting profiles in the presence of saturating fluorescent DNA binding dyes.

ANTI-SILENCING FUNCTION1 proteins are involved in ultraviolet-induced DNA damage repair and are cell cycle regulated by E2F transcription factors in Arabidopsis.

ANTI-SILENCING FUNCTION1 (ASF1) is a key histone H3/H4 chaperone that participates in a variety of DNA- and chromatin-related processes, including DNA repair, where chromatin assembly and disassembly are of primary relevance. Information concerning the role of ASF1 proteins in the post-ultraviolet (UV) response in higher plants is currently limited. In Arabidopsis (Arabidopsis thaliana), an initial analysis of in vivo localization of ASF1A and ASF1B indicates that both proteins are mainly expressed in proliferative tissues. In silico promoter analysis identified ASF1A and ASF1B as potential targets of E2F corresponds to Adenovirus E2 Binding Factor. [corrected]. These observations were experimentally validated, both in vitro, by electrophoretic mobility shift assays, and in vivo, by chromatin immunoprecipitation assays and expression analysis using transgenic plants with altered levels of different E2F transcription factors. These data suggest that ASF1A and ASF1B are regulated during cell cycle progression through E2F transcription factors. In addition, we found that ASF1A and ASF1B are associated with the UV-B-induced DNA damage response in Arabidopsis. Transcript levels of ASF1A and ASF1B were increased following UV-B treatment. Consistent with a potential role in UV-B response, RNA interference-silenced plants of both genes showed increased sensitivity to UV-B compared with wild-type plants. Finally, by coimmunoprecipitation analysis, we found that ASF1 physically interacts with amino-terminal acetylated histones H3 and H4 and with acetyltransferases of the Histone Acetyl Transferase subfamily, which are known to be involved in cell cycle control and DNA repair, among other functions. Together, we provide evidence that ASF1A and ASF1B are regulated by cell cycle progression and are involved in DNA repair after UV-B irradiation.

Mismatch recognition function of Arabidopsis thaliana MutSγ.

Genetic stability depends in part on an efficient DNA lesion recognition and correction by the DNA mismatch repair (MMR) system. In eukaryotes, MMR is initiated by the binding of heterodimeric MutS homologue (MSH) complexes, MSH2-MSH6 and MSH2-MSH3, which recognize and bind mismatches and unpaired nucleotides. Plants encode another mismatch recognition protein, named MSH7. MSH7 forms a heterodimer with MSH2 and the protein complex is designated MutSγ. We here report the effect the expression of Arabidopsis MSH2 and MSH7 alone or in combination exert on the genomic stability of Saccharomyces cerevisiae. AtMSH2 and AtMutSγ proteins failed to complement the hypermutator phenotype of an msh2 deficient strain. However, overexpressing AtMutSγ in MMR proficient strains generated a 4-fold increase in CAN1 forward mutation rate, when compared to wild-type strains. Can(r) mutation spectrum analysis of AtMutSγ overproducing strains revealed a substantial increase in the frequency of base substitution mutations, including an increased accumulation of base pair changes from G:C to A:T and T:A to C:G, G:C or A:T. Taken together, these results suggest that AtMutSγ affects yeast genomic stability by recognizing specific mismatches and preventing correction by yeast MutSα and MutSß, with subsequent inability to interact with yeast downstream proteins needed to complete MMR.

Yeast mutator phenotype enforced by Arabidopsis PMS1 expression.

The DNA mismatch repair (MMR) system is a major DNA repair pathway whose function is critical for the correction of DNA biosynthetic errors. MMR is initiated by the binding of MutS proteins to mismatches and unpaired nucleotides followed by the recruitment of MutL proteins. The major MutL activity in eukaryotes is performed by MutLα, the heterocomplex of MLH1-PMS1 in yeast and plants and MLH1-PMS2 in humans. We here report the effect the expression of Arabidopsis PMS1 protein exerts on Saccharomyces cerevisiae genomic stability. A strain carrying specific microsatellite instability reporter systems was chosen for the study. The plant protein failed to complement the hypermutator phenotype of a pms1 deficient strain but increased approximately 14-fold and 2,000-fold the mutation rates of his7-2 and lys2::InsE-A 14 loci of MMR proficient strains when compared to wild-type strains, respectively. Overexpressing AtMLH1 in the AtPMS1-overproducing strain generated an increase in mutation rate comparable to that of AtPMS1 expression alone. Deletion of the C-terminal residues implicated in protein-protein interaction and including the putative endonuclease sequence of AtPMS1 completely eliminated the mutator phenotype. Taken together, these results indicate that the plant proteins affect yeast genomic stability, very possibly altering protein-protein interactions that are necessary to complete repair.

Research on plants for the understanding of diseases of nuclear and mitochondrial origin.

Different model organisms, such as Escherichia coli, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, mouse, cultured human cell lines, among others, were used to study the mechanisms of several human diseases. Since human genes and proteins have been structurally and functionally conserved in plant organisms, the use of plants, especially Arabidopsis thaliana, as a model system to relate molecular defects to clinical disorders has recently increased. Here, we briefly review our current knowledge of human diseases of nuclear and mitochondrial origin and summarize the experimental findings of plant homologs implicated in each process.