Two high-mobility group domains of MHG1 are necessary to maintain mtDNA in Neurospora crassa.

The mhg1 (NCU02695/ada-23) gene encodes the mitochondrial high-mobility group box (HMG-box or HMGB) protein in Neurospora crassa. The mhg1 KO strain (mhg1KO) has mitochondrial DNA (mtDNA) instability and a short lifespan; however, the function of MHG1 remains unclear. To investigate the role of this protein in the maintenance of mtDNA, domain deleted MHG1 proteins were expressed in the mhg1KO strain, and their effects were analyzed. We identified two putative HMG-domains, HMGBI and HMGBII. Although deletion of the HMG-box did not abolish MHG1's mitochondrial localization, the mhg1KO phenotype of a severe growth defect and a high sensitivity to mutagens could not be restored by introduction of HMG-box deleted mhg1 gene into the KO strain. It was indicated that recombinant full-length MHG1, i.e., mitochondrial targeting sequence (MTS) containing protein, did not exhibit explicit DNA binding, whereas the MHG1 protein truncated for the MTS sequence did in vitro by an electrophoretic mobility shift assay. Furthermore, recombinant MHG1 protein lacking MTS and HMG-domains, either HMGBI or HMGBII, had DNA affinity and an altered band shift pattern compared with MTS-truncated MHG1 protein. These results suggest that cleavage of MTS and appropriate DNA binding via HMG-domains are indispensable for maintaining mtDNA in N. crassa.

A20/AN1 zinc-finger proteins positively regulate major latex-like proteins, transporting factors toward dioxin-like compounds in Cucurbita pepo.

The Cucurbitaceae family accumulates dioxin-like compounds in its fruits. We previously showed that A20/AN1 zinc finger protein (ZFP) genes were highly expressed in the zucchini (Cucurbita pepo) subspecies pepo, which accumulates dioxin-like compounds at high concentrations. Transgenic tobacco (Nicotiana tabacum) plants overexpressing A20/AN1 ZFP genes show accumulation of dioxin-like compounds in their upper parts. However, the mechanisms underlying the accumulation of dioxin-like compounds regulated by the A20/AN1 ZFPs remain unclear. Here, we show that A20/AN1 ZFPs positively regulate the expression of the major latex-like protein (MLP) and its homolog genes in N. tabacum and C. pepo. MLPs are involved in the transport of dioxin-like compounds from the roots to the upper parts of C. pepo. Overexpression of A20/AN1 ZFP genes in N. tabacum leads to the upregulation of pathogenesis-related protein class-10 genes with the binding ability toward dioxin-like compounds. Our results demonstrated that A20/AN1 ZFPs upregulate MLP and its homolog genes in N. tabacum and C. pepo, resulting in the accumulation of dioxin-like compounds.

MLP-PG1, a major latex-like protein identified in Cucurbita pepo, confers resistance through the induction of pathogenesis-related genes.

MAIN CONCLUSION: MLP-PG1, identified in Cucurbita pepo, plays a crucial role in resistance against fungal pathogens through the induction of pathogenesis-related genes. ASTRACT: MLP-PG1, a major latex-like protein (MLP) from zucchini (Cucurbita pepo), was identified as a transporting factor for hydrophobic organic pollutants. MLPs are members of the Bet v 1 family, similar to pathogenesis-related class 10 proteins (PR-10s). However, the biological functions of MLPs remain unclear. Herein, we show that MLP-PG1 induces the expression of pathogenesis-related (PR) genes and indirectly promotes resistance against pathogens. The activity of the MLP-PG1 promoter in leaves of transgenic tobacco plants was significantly enhanced by inoculation with Pseudomonas syringae pv. tabaci. However, MLP-PG1 did not induce direct resistance through RNase activity. Therefore, we examined the possibility that MLP-PG1 is indirectly involved in resistance; indeed, we found that MLP-PG1 induced the expression of defense-related genes. Overexpression of MLP-PG1 highly upregulated PR-2 and PR-5 and decreased the area of lesions caused by Botrytis cinerea in the leaves of transgenic tobacco plants. Our results demonstrate that MLP-PG1 is involved in indirect resistance against plant diseases, especially caused by fungal pathogens, through the induction of PR genes. This study is the first report to show the induction of PR genes by the expression of MLP from the RNA sequencing analysis and the involvement of MLP-PG1 in the resistance.

A partial photoreactivation defect phenotype is not due to unrepaired ultraviolet-induced pyrimidine dimers in ultraviolet-sensitive mutants of Neurospora crassa.

Photoreactivation is a mechanism in which photolyase directly repairs either cyclobutane pyrimidine dimers (CPDs) or (6-4) photoproducts [(6-4) PPs] caused by ultraviolet (UV) light. In the filamentous fungus Neurospora crassa, some UV-sensitive mutants such as mus-44 have been reported to exhibit a partial photoreactivation defect (PPD) phenotype, but its mechanism has not been elucidated for a long time. In this study, the N. crassa CPD photolyase PHR was overexpressed in the Δmus-44 strain, but photoreactivation ability was not increased. Furthermore, Escherichia coli CPD photolyase or Arabidopsis thaliana (6-4) PP photolyase was also introduced into Δmus-44; however, the PPD phenotype was not complemented. These results suggested that the PPD phenotype in N. crassa is not caused by residual unrepaired pyrimidine dimers, which are the main type of DNA damage caused by UV irradiation. Finally, we revealed that Δmus-44, but not the Δmus-43 strain, which does not show the PPD phenotype, displayed higher sensitivity with increasing dose rate of UV. Moreover, Δmus-44 was also sensitive to an interstrand crosslinking agent. This indicates that the high dose of UV in our experimental condition induces DNA damage other than pyrimidine dimers, and that such damage is a likely cause of the PPD phenotype.

Suppression of the genes responsible for transporting hydrophobic pollutants leads to the production of safer crops.

Hydrophobic pollutants have become widely distributed across the world. From an agricultural perspective, their accumulation in crops from contaminated soil threatens food security and quality, leading to many diseases in humans. The Cucurbitaceae family can accumulate high concentrations of hydrophobic pollutants in their aerial parts. The Cucurbitaceae family contains major latex-like proteins (MLPs) as transporting factors for hydrophobic pollutants. MLP genes are expressed in the roots in which the MLPs bind hydrophobic pollutants. MLPs transport these hydrophobic pollutants to the aerial parts of the plant through the xylem vessels. As a result, hydrophobic pollutant contamination occurs in the Cucurbitaceae family. In this study, we suppressed the expression of MLP genes in the roots and reduced the amounts of MLPs with pesticide treatments. First, the fungicides Benlate and Daconil that deceased the hydrophobic pollutant, perylene, concentration in the xylem sap of zucchini plants were selected. Daconil suppressed the transcription activity of MLP in the roots. In the Daconil treatment, the amount of MLPs in the roots and xylem sap of zucchini plants was decreased, and the concentrations of the hydrophobic pollutants, pyrene and dieldrin, were significantly decreased. Our research contributes to the production of safer crops.

The msh1 gene is responsible for short life span mutant natural death and functions to maintain mitochondrial DNA integrity.

Wild-type filamentous fungus Neurospora crassa continues to grow its hyphae for a very lengthy period of time (>2 years), whereas mutations at the natural death (nd) locus shorten life span (approximately 20 days). By positional cloning based on heat augmented mutagen sensitivity of the nd strain, we identified a nonsense mutation in the msh1 gene, an eukaryotic homolog of bacterial MutS, and this mutation resulted in encoding non-functional polypeptide. By tagging with GFP, subcellular localization of the MSH1 protein in the mitochondria was observed, and knock out of the msh1 gene caused severe growth deficiency accompanying mitochondrial DNA (mtDNA) aberrations such as large-scale mtDNA deletions and rearrangements as seen in the nd strain. These results suggested that MSH1 may maintain mtDNA integrity. Thus, loss of function compromises mtDNA, leading to the acceleration of cellular aging.

Effects of Arabidopsis Ku80 deletion on the integration of the left border of T-DNA into plant chromosomal DNA via Agrobacterium tumefaciens.

T-DNA integration into plant chromosomal DNA via Agrobacterium tumefaciens can be achieved by exploiting the double-strand break repair system of the host's DNA. However, the detailed mechanism of T-DNA integration remains unclear. Here, a sequence analysis of the junction sequences of T-DNA and chromosomal DNA was performed to assess the mechanism of T-DNA integration. T-DNA was introduced into Arabidopsis wild-type and NHEJ-deficient ku80 mutant plants using the floral dip method; the junctions of the left border (LB) of T-DNA were subsequently analyzed by adapter PCR. The most frequent junction of the LB of T-DNA with chromosomal DNA was of the filler DNA type in both lines. The lengths of direct or inverted repeat sequences within or around the filler DNA sequence were greater in the ku80 mutant. In addition, the frequency of T-DNA integration near a transcription start site was significantly higher in the ku80 mutant. Our observations suggest that the presence of the Ku80 protein affects the location of the integration of T-DNA and the pattern of formation of repeat sequences within or around the filler DNA during LB integration into chromosomal DNA.

Analysis of localization of cell-cycle regulators in Neurospora crassa.

Most fungi are multinucleated organisms. In some fungi, they have asynchronous nuclei in the same cytoplasm. We analyzed a cell-cycle regulation mechanism using a model fungus Neurospora crassa, which can make heterokaryon cells. G1/S cyclin CLN-1 and cyclin-dependent kinase CDC-2 were tagged with different fluorescence in different strains and expressed. By forming a heterokaryon strain of these, two different fluorescence-tagged proteins were expressed in the same cytoplasm. CDC-2 was localized in all nuclei, whereas CLN-1 was not detected in most of the nuclei and was dispersed in the cytoplasm with small granular clusters. This indicates that in multinucleated fungi, cell-cycle regulators, similar to other proteins, are shared around the nuclei regardless of different cell-cycle stages. Moreover, each nucleus can select and use a special cell-cycle regulator only when it is necessary. Fungal nuclei may have a novel pickup mechanism of necessary proteins from their cytoplasm at the point of use.

Phenotypic analysis of newly isolated short-lifespan Neurospora crassa mutant deficient in a high mobility group box protein.

To elucidate genetic mechanisms affecting the lifespan of the filamentous fungus Neurospora crassa, we attempted to identify a gene of which a defect causes a short-lifespan. By screening a Neurospora knockout library, provided by the Fungal Genetics Stock Center at Kansas State University, several KO strains with a short-lifespan were isolated. FGSC#11693 is one of these, which shows similar phenotypes to known Neurospora short-lifespan mutants as follows: 1) hyphal growth ceases after about 2weeks of cultivation, despite that of the wild-type continuing for over 2years, 2) viability of conidia is lower than that of the wild-type, and 3) high sensitivity to mutagens such as methyl methanesulfonate, ultraviolet radiation, and hydroxyl urea is exhibited. The NCU number of the knocked-out gene in the KO strain is NCU02695, and recovery from the short-lifespan and mutagen sensitivity was achieved by the introduction of this gene from the wild-type. The putative amino acid sequence of the knocked-out gene contains two high mobility group box domains and a mitochondrial localization signal is found at the N-terminal of this sequence. Upon analyzing the subcellular localization of the gene product fused with GFP, GFP signals were detected in mitochondria. From these observations, the gene and KO strain were named mitochondrial high mobility group box protein 1 (MHG1) and mhg1KO strain, respectively. The amount of mtDNA relative to the nuclear amount was lower in the mhg1KO strain than in the wild-type. mtDNA aberration was also observed in the mhg1KO strain. These results suggest that the MHG1 protein plays an important role in the maintenance of mitochondrial DNA, and mitochondrial abnormality caused by mtDNA aberration is responsible for the short-lifespan of the mhg1KO strain.

Zinc finger protein genes from Cucurbita pepo are promising tools for conferring non-Cucurbitaceae plants with ability to accumulate persistent organic pollutants.

Some cultivars of cucumbers, melons, pumpkins, and zucchini, which are members of the Cucurbitaceae family, are uniquely subject to contamination by hydrophobic pollutants such as the organohalogen insecticides DDT. However, the molecular mechanisms for the accumulation of these pollutants in cucurbits have not been determined. Here, cDNA subtraction analysis of Cucurbita pepo cultivars that are low and high accumulators of hydrophobic contaminants revealed that a gene for zinc finger proteins (ZFPs) are preferentially expressed in high accumulators. The cloned CpZFP genes were classified into 2 types: (1) the PBG type, which were expressed in C. pepo cultivars Patty Green, Black Beauty, and Gold Rush, and (2) the BG type, which were expressed in Black Beauty and Gold Rush. Expression of these CpZFP genes in transgenic tobacco plants carrying an aryl hydrocarbon receptor-based inducible gene expression system significantly induced ß-glucuronidase activity when the plants were treated with a polychlorinated biphenyl (PCB) compound, indicating that highly hydrophobic PCBs accumulated in the plants. In transgenic tobacco plants carrying CpZFPs, accumulation of dioxins and dioxin-like compounds increased in their aerial parts when they were cultivated in the dioxin-contaminated soil. In summary, we propose that addition of CpZFP genes is a promising tool for conferring noncucurbits with the ability to accumulate hydrophobic contaminants.

Mutational effects of γ-rays and carbon ion beams on Arabidopsis seedlings.

To assess the mutational effects of radiation on vigorously proliferating plant tissue, the mutation spectrum was analyzed with Arabidopsis seedlings using the plasmid-rescue method. Transgenic plants containing the Escherichia coli rpsL gene were irradiated with γ-rays and carbon ion beams (320-MeV (12)C(6+)), and mutations in the rpsL gene were analyzed. Mutant frequency increased significantly following irradiation by γ-rays, but not by 320-MeV (12)C(6+). Mutation spectra showed that both radiations increased the frequency of frameshifts and other mutations, including deletions and insertions, but only γ-rays increased the frequency of total base substitutions. These results suggest that the type of DNA lesions which cause base substitutions were less often induced by 320-MeV (12)C(6+) than by γ-rays in Arabidopsis seedlings. Furthermore, γ-rays never increased the frequencies of G:C to T:A or A:T to C:G transversions, which are caused by oxidized guanine; 320-MeV (12)C(6+), however, produced a slight increase in both transversions. Instead, γ-rays produced a significant increase in the frequency of G:C to A:T transitions. These results suggest that 8-oxoguanine has little effect on mutagenesis in Arabidopsis cells.

Arabidopsis sos1 mutant in a salt-tolerant accession revealed an importance of salt acclimation ability in plant salt tolerance.

An analysis of the salinity tolerance of 354 Arabidopsis thaliana accessions showed that some accessions were more tolerant to salt shock than the reference accession, Col-0, when transferred from 0 to 225 mM NaCl. In addition, several accessions, including Zu-0, showed marked acquired salt tolerance after exposure to moderate salt stress. It is likely therefore that Arabidopsis plants have at least two types of tolerance, salt shock tolerance and acquired salt tolerance. To evaluate a role of well-known salt shock tolerant gene SOS1 in acquired salt tolerance, we isolated a sos1 mutant from ion-beam-mutagenized Zu-0 seedlings. The mutant showed severe growth inhibition under salt shock stress owing to a single base deletion in the SOS1 gene and was even more salt sensitive than Col-0. Nevertheless, it was able to survive after acclimation on 100 mM NaCl for 7 d followed by 750 mM sorbitol for 20 d, whereas Col-0 became chlorotic under the same conditions. We propose that genes for salt acclimation ability are different from genes for salt shock tolerance and play an important role in the acquisition of salt or osmotic tolerance.

Mutagenic effects of carbon ions near the range end in plants.

To gain insight into the mutagenic effects of accelerated heavy ions in plants, the mutagenic effects of carbon ions near the range end (mean linear energy transfer (LET): 425keV/µm) were compared with the effects of carbon ions penetrating the seeds (mean LET: 113keV/µm). Mutational analysis by plasmid rescue of Escherichia coli rpsL from irradiated Arabidopsis plants showed a 2.7-fold increase in mutant frequency for 113keV/µm carbon ions, whereas no enhancement of mutant frequency was observed for carbon ions near the range end. This suggested that carbon ions near the range end induced mutations that were not recovered by plasmid rescue. An Arabidopsis DNA ligase IV mutant, deficient in non-homologous end-joining repair, showed hyper-sensitivity to both types of carbon-ion irradiation. The difference in radiation sensitivity between the wild type and the repair-deficient mutant was greatly diminished for carbon ions near the range end, suggesting that these ions induce irreparable DNA damage. Mutational analysis of the Arabidopsis GL1 locus showed that while the frequency of generation of glabrous mutant sectors was not different between the two types of carbon-ion irradiation, large deletions (>∼30kb) were six times more frequently induced by carbon ions near the range end. When 352keV/µm neon ions were used, these showed a 6.4 times increase in the frequency of induced large deletions compared with the 113keV/µm carbon ions. We suggest that the proportion of large deletions increases with LET in plants, as has been reported for mammalian cells. The nature of mutations induced in plants by carbon ions near the range end is discussed in relation to mutation detection by plasmid rescue and transmissibility to progeny.

Mutational effects of different LET radiations in rpsL transgenic Arabidopsis.

PURPOSE: In an effort to assess the characteristics of mutation induced by different linear energy transfer (LET) radiation in higher plants, the mutational effects of carbon-ion beams and gamma-rays were investigated in Arabidopsis. MATERIALS AND METHODS: The rpsL (Escherichia coli ribosomal protein small subunit S12) transgenic Arabidopsis (Arabidopsis/rpsL) mutation detection system was adopted. Dry seeds of Arabidopsis/rpsL were irradiated with gamma-rays and 208-MeV carbon ions (208-MeV (12)C(5+)), and the mutation frequency and mutation spectrum were examined. RESULTS: The frequency of mutant clones increased following irradiation with 208-MeV (12)C(5+) and gamma-rays. Mutation spectrum analysis showed that G:C to A:T transitions and >2 bp deletions/insertions were significantly induced by both 208-MeV (12)C(5+) and gamma-rays. -1 and -2 frameshift mutations were characteristic in the gamma-ray irradiated group. CONCLUSIONS: 208-MeV (12)C(5+) and gamma-rays induced different intragenic mutations in respect to the size of deletions, reflecting differences in the nature of the DNA damage induced. Our results also suggested that base substitutions derived from the generation of 8-oxoguanine were low in dry seeds. The mutation spectrum obtained in this study might have reflected the characteristic conditions of plant dry seeds such as low water content and cell proliferation activity.

A new system for detecting mutations in arabidopsis thaliana and the mutational spectra resulting from ethylmethanesulfonate treatment.

A system was developed for the detection and analysis of mutations occurring on chromosomal DNA in plants. The plasmid pML4, carrying the Escherichia coli rpsL gene, a target gene for mutagenesis, was inserted into a shuttle vector, pCGN5138, to construct a plasmid which could be used for the transformation of plants. pML4 sequences were introduced into Arabidopsis thaliana mediated by Agrobacterium. The pML4 DNA was rescued from transgenic Arabidopsis plants exposed to mutagens, and the plasmids were introduced into Escherichia coli DH10B to isolate mutant clones. In this system, any form of inactivation mutation in the rpsL gene can be positively selected since it makes the E. coli cells resistant to streptomycin. Here we report that the system could detect the mutagenic effect of ethylmethanesulfonate (EMS). Further characterization of the mutants revealed that G:C to A:T transitions predominated among the EMS-induced mutations. This assay system is useful for the detection and analysis of mutations arising on chromosomal DNA in plants, and should be useful for evaluating analysis of the effects of environmental mutagens.

CPD photolyase gene from Spinacia oleracea: repair of UV-damaged DNA and expression in plant organs.

The UV-B radiation contained in solar radiation has deleterious effects on plant growth, development and physiology. Specific damage to DNA caused by UV radiation involves the cyclobutyl pyrimidine dimers (CPD) and the pyrimidine (6-4) pyrimidone photoproducts. CPDs are repaired by CPD photolyase via a UV-A/blue light-dependent mechanism. The gene for the class II CPD photolyase has been cloned from higher plants such as Arabidopsis, cucumbers and rice. We isolated and characterized the cDNA and a genomic clone encoding the spinach class II CPD photolyase. The gene consisted of 3777 bases and 9 exons. The sequence of amino acids predicted from the nucleotide sequence of the cDNA of the gene was highly homologous to that of the higher plants listed above. When a photolyase-deficient Escherichia coli strain was transformed with the cDNA, photoreactivation activity was partially restored, by the illumination with photoreactivating light, resulting in an increased survival and decreased content of CPDs in the Escherichia coli genome. In both the male and female plants, the gene was highly expressed in leaves and flowers under the condition of 14-h light and 10-h dark cycle. The expression in the roots was quite low compared with the other organs.