Functional stacking of three resistance genes against Phytophthora infestans in potato

Functional stacking of broad spectrum resistance (R) genes could potentially be an effective strategy for more durable disease resistance, for example, to potato late blight caused by Phytophthora infestans (Pi). For this reason, three broad spectrum potato R genes (Rpi), Rpi-sto1 (Solanum stoloniferum), Rpi-vnt1.1 (S. venturii) and Rpi-blb3 (S. bulbocastanum) were selected, combined into a single binary vector pBINPLUS and transformed into the susceptible cultivar Desiree. Among the 550 kanamycin resistant regenerants, 28 were further investigated by gene specific PCRs. All regenerants were positive for the nptII gene and 23 of them contained the three Rpi genes, referred to as triple Rpi gene transformants. Detached leaf assay and agro-infiltration of avirulence (Avr) genes showed that the 23 triple Rpi gene transformants were resistant to the selected isolates and showed HR with the three Avr effectors indicating functional stacking of all the three Rpi genes. It is concluded that Avr genes, corresponding to the R genes to be stacked, must be available in order to assay for functionality of each stack component. No indications were found for silencing or any other negative effects affecting the function of the inserted Rpi genes. The resistance spectrum of these 23 triple Rpi gene transformants was, as expected, a sum of the spectra from the three individual Rpi genes. This is the first example of a one-step approach for the simultaneous domestication of three natural R genes against a single disease by genetic transformation.     

Growth requirements of some fungi causing maduromycosis

Three strains ofMadurella mycetomi, two ofM. grisea, and two ofRhinocladiella mansonii have been studied for possible differences in growth requirements which might be used for distinguishing these species. Under the experimental conditions, an incubation temperature of 37C suitedM. mycetomi about as well as 30C.R. mansonii grew less well at 37C than at 30C, andM. grisea did not grow at the higher temperature. M. grisea andR. mansonii further differed fromM. mycetomi in that they required thiamine for growth. The pH tolerance of all the strains was very wide. Asparagine and potassium nitrate were readily utilized by all the strains, but ammonium salts were not. Urea was poorly used byM. mycetomi; the other species did not use it. A possible relationship ofM. grisea andR. mansonii is discussed.     

NUT1, a major nitrogen regulatory gene inMagnaporthe grisea, is dispensable for pathogenicity

NUT1, a gene homologous to the major nitrogen regulatory genesnit-2 ofNeurospora crassa andareA ofAspergillus nidulans, was isolated from the rice blast fungus,Magnaporthe grisea. NUT1 encodes a protein of 956 amino acid residues and, likenit-2 andareA, has a single putative zinc finger DNA-binding domain. Functional equivalence ofNUT1 toareA was demonstrated by introducing theNUT1 gene by DNA-mediated transformation into anareA loss-of-function mutant ofA. nidulans. The introducedNUT1 gene fully complemented theareA null mutation, restoring to the mutant the ability to utilize a variety of nitrogen sources. In addition, the sensitivity ofAspergillus NUT1 transformants to ammonium repression of extracellular protease activity was comparable to that of wild-typeA. nidulans. Thus,NUT1 andareA encode functionally equivalent gene products that activate expression of nitrogen-regulated genes. A one-step gene disruption strategy was used to generatenutl ^– mutants ofM. grisea by transforming a rice-infecting strain with a disruption vector in which a gene for hygromycin B phosphotransferase (Hyg) replaced the zinc-finger DNA-binding motif ofNUT1. Of 31 hygromycin B (hyg B)-resistant transformants shown by Southern hybridization to contain a disruptedNUT1 gene (nut1::Hyg), 26 resulted from single-copy replacement events at theNUT1 locus. Althoughnut1 ^– transformants ofM. grisea failed to grown on a variety of nitrogen sources, glutamate, proline and alanine could still be utilized. This contrasts withA. nidulans where disruption of the zinc-finger region ofareA prevents utilization of nitrogen sources other than ammonium and glutamine. The role ofNUT1 and regulation of nitrogen metabolism in the disease process was evaluated by pathogenicity assays. The infection efficiency ofnut1 ^– transformants on susceptible rice plants was similar to that of the parental strain, although lesions were reduced in size. These studies demonstrate that theM. grisea NUT1 gene activates expression of nitrogen-regulated genes but is dispensable for pathogenicity.     

Isolation of an Rx homolog from C. annuum and the evolution of Rx genes in the Solanaceae family

The well-conserved NBS domain of resistance (R) genes cloned from many plants allows the use of a PCR-based approach to isolate resistance gene analogs (RGAs). In this study, we isolated an RGA (CapRGC) from Capsicum annuum “CM334” using a PCR-based approach. This sequence encodes a protein with very high similarity to Rx genes, the Potato Virus X (PVX) R genes from potato. An evolutionary analysis of the CapRGC gene and its homologs retrieved by an extensive search of a Solanaceae database provided evidence that Rx-like genes (eight ESTs or genes that show very high similarity to Rx) appear to have diverged from R1 [an NBS-LRR R gene against late blight (Phytophthora infestans) from potato]-like genes. Structural comparison of the NBS domains of all the homologs in Solanaceae revealed that one novel motif, 14, is specific to the Rx-like genes, and also indicated that several other novel motifs are characteristic of the R1-like genes. Our results suggest that Rx-like genes are ancient but conserved. Furthermore, the novel conserved motifs can provide a basis for biochemical structural–function analysis and be used for degenerate primer design for the isolation of Rx-like sequences in other plant species. Comparative mapping study revealed that the position of CapRGC is syntenic to the locations of Rx and its homolog genes in the potato and tomato, but cosegregation analysis showed that CapRGC may not be the R gene against PVX in pepper. Our results confirm previous observations that the specificity of R genes is not conserved, while the structure and function of R genes are conserved. It appears that CapRGC may function as a resistance gene to another pathogen, such as the nematode to which the structure of CapRGC is most similar.     

Activation and analysis of crypticcrt genes for carotenoid biosynthesis fromStreptomyces griseus

Genes encoding enzymes with sequence similarity to carotenoid biosynthetic enzymes of other organisms were cloned fromStreptomyces griseus JA3933 and transformed into the colourless (non-daunorubicin producing) mutantStreptomyces griseus IMET JA3933/956/2. Cells harbouring these genes showed an orange-red pigmentation, caused by the strongly hydrophobic, membrane-bound lycopene. The cloned fragment (9 kb) contained seven genes, four transcribed in one direction (crtEIBV) and three (crtYTU) transcribed convergently to them. Three of these genes encode polypeptides that resemble geranylgeranyl-pyrophosphate (GGPP) synthases (CrtE), phytoene synthases (PS) (CrtB) and phytoene dehydrogenases (PDH) (CrtI), respectively, of various bacteria. These enzymes are sufficient for the formation of lycopene.crtE alone was sufficient to induce zeaxanthin formation in anEscherichia coli clone containing thecrt gene cluster fromErwinia herbicola deleted forcrtE. The combination ofcrtE andcrtB led to formation of phytoene inS. griseus. The putativecrtEp promoter region was cloned and mapped by primer extension analysis. In a gel retardation experiment, this fragment was specifically shifted by an unknown protein. CrtY shows similarity to lycopene cyclases that convert lycopene into-carotene, CrtT resembles various methyltransferases and CrtU a dehydrogenase. We conclude that these genes are functionally intact, but not expressed (cryptic) in the wild-typeS. griseus strain.     

Cloning and characterization of the lactate dehydrogenase genes from<Emphasis Type="Italic">Lactobacillus</Emphasis> sp. RKY2

Lactic acid is an environmentally benign organic acid that could be used as a raw material for biodegradable plastics if it can be inexpensively produced by fermentation. Two genes (IdhL andIdhD) encoding the L-(+) and D-(−) lactate dehydrogenases (L-LDH and D-LDH) were cloned fromLactobacillus sp., RKY2, which is a lactic acid hyper-producing bacterium isolated from Kimchi. Open reading frames ofIdhL for andIdhD for the L and D-LDH genes were 962 and 998 bp, respectively. Both the L(+)- and D(−)-LDH proteins showed the highest degree of homology with the L- and D-lactate dehydrogenase genes ofLactobacillus plantarum. The conserved residues in the catalytic activity and substrate binding of both LDHs were identified in both enzymes.     

Virus-induced silencing of WIPK and SIPK genes reduces resistance to a bacterial pathogen,but has no effect on the INF1-induced hypersensitive response (HR) in Nicotiana benthamiana

Activation of two mitogen-activated protein kinases (MAPKs), wound-induced protein kinase (WIPK) and salicylic acid-induced protein kinase (SIPK), is one of the earliest responses that occur in tobacco plants that have been wounded, treated with pathogen-derived elicitors or challenged with avirulent pathogens. We isolated cDNAs for these MAPKs ( NbWIPK and NbSIPK) from Nicotiana benthamiana. The function of NbWIPK and NbSIPK in mediating the hypersensitive response (HR) triggered by infiltration with INF1 protein (the major elicitin secreted by Phytophthora infestans), and the defense response to an incompatible bacterial pathogen ( Pseudomonas cichorii), was investigated by employing virus-induced gene silencing (VIGS) to inhibit expression of the WIPK and SIPK genes in N. benthamiana. Silencing of WIPK or SIPK, or both genes simultaneously, resulted in reduced resistance to P. cichorii, but no change was observed in the timing or extent of HR development after treatment with INF1.Communicated by R. G. Herrmann     

Drosophila virilis histone gene clusters lacking H1 coding segments

Summary Approximately 30–40% ofDrosophila virilis DNA complementary to clonedDrosophila histone genes is reduced to 3.4-kilobase-pair (kbp) segments by Bgl I or Bgl II digestion. The core histone genes of a 3.4-kbp Bgl II segment cloned in the plasmid pDv3/3.4 have the same order as theD. melanogaster core histone genes in the plasmid cDm500: . Nonetheless, pDv3/3.4 and cDm500 have different histone gene configurations: In pDv3/3.4, the region between the H2B and H3 genes contains 0.35 kbp and cannot encode histone H1; in cDm500, the region contains 2.0 kbp and encodes histone H1. The lack of an H1 gene between the H2B and H3 genes in 30–40% ofD. virilis histone gene clusters suggests that changes in histone gene arrays have occurred during the evolution ofDrosophila. The ancestors of modernDrosophila may have possessed multiple varieties of histone gene clusters, which were subsequently lost differentially in thevirilis andmelanogaster lineages. Alternatively, they may have possessed a single variety, which was rearranged during evolution. The H1 genes ofD. virilis andD. melanogaster did not cross-hybridize in vitro under conditions that maintain stable duplexes between DNAs that are 75% homologous. Consequently,D. virilis H1 genes could not be visualized by hybridization to an H1-specific probe and thus remain unidentified. Our observations suggest that the coding segments in the H1 genes ofD. virilis andD. melanogaster are >25% divergent. Our estimate of sequence divergence in the H1 genes ofD. virilis andD. melanogaster seems high until one considers that the coding sequences of cloned H1 genes from the closely related speciesD. melanogaster andD. simulans are 5% divergent.     

Diversity of arbuscular mycorrhizal fungi colonising roots of the grass species<Emphasis Type="Italic"> Agrostis capillaris</Emphasis> and<Emphasis Type="Italic"> Lolium perenne</Emphasis> in a field experiment

Analysis of arbuscular mycorrhizal (AM) fungal diversity through morphological characters of spores and intraradicular hyphae has suggested previously that preferential associations occur between plants and AM fungi. A field experiment was established to investigate whether AM fungal diversity is affected by different host plants in upland grasslands. Indigenous vegetation from plots in an unimproved pasture was replaced with monocultures of either Agrostis capillaris or Lolium perenne. Modification of the diversity of AM fungi in these plots was evaluated by analysis of partial sequences in the large subunit (LSU) ribosomal RNA (rDNA) genes. General primers for AM fungi were designed for the PCR amplification of partial sequences using DNA extracted from root tissues of A. capillaris and L. perenne. PCR products were used to construct LSU rDNA libraries. Sequencing of randomly selected clones indicated that plant roots were colonised by AM fungi belonging to the genera Glomus, Acaulospora and Scutellospora. There was a difference in the diversity of AM fungi colonising roots of A. capillaris and L. perenne that was confirmed by PCR using primers specific for each sequence group. These molecular data suggest the existence of a selection pressure of plants on AM fungal communities.     

Cloning, Sequencing, and Transgenic Expression ofPodospora curvicollaandSordaria macrosporaeEF1A Genes: Relationship between Cytosolic Translation and Longevity in Filamentous Fungi

We have cloned and sequenced the gene encoding the translation elongation factor eEF1A from two filamentous fungi,Podospora curvicollaandSordaria macrospora.These fungi are close relatives ofPodospora anserinaand also show senescence syndromes. Comparison of the sequences of the deduced proteins with that ofP. anserinareveals that the three proteins differ in several positions. Replacement of theP. anserinagene by either of the two exogenous genes does not entail any modification inP. anserinaphysiology; the longevity of the fungus is not affected. No alteration ofin vivotranslational accuracy was detected; however, the exogenous proteins nonetheless promoted a modification of the resistance to the aminoglycoside antibiotic paromomycin. These data suggest that optimization of life span between these closely related fungi has likely not been performed during evolution through modifications of eEF1A activity, despite the fact that mutations in this factor can drastically affect longevity.     

cDNA Cloning and Gene Mapping of Human Homologs forSchizosaccharomyces pombe rad17, rad1,andhus1and Cloning of Homologs from Mouse,Caenorhabditis elegans,andDrosophila melanogaster

Mutations in DNA repair/cell cycle checkpoint genes can lead to the development of cancer. The cloning of human homologs of yeast DNA repair/cell cycle checkpoint genes should yield candidates for human tumor suppressor genes as well as identifying potential targets for cancer therapy. TheSchizosaccharomyces pombegenesrad17, rad1,andhus1have been identified as playing roles in DNA repair and cell cycle checkpoint control pathways. We have cloned the cDNA for the human homolog ofS. pombe rad17,RAD17, which localizes to chromosomal location 5q13 by fluorescencein situhybridization and radiation hybrid mapping; the cDNA for the human homolog ofS. pombe rad1,RAD1, which maps to 5p14–p13.2; and the cDNA for the human homolog ofS. pombe hus1,HUS1, which maps to 7p13–p12. The human gene loci have previously been identified as regions containing tumor suppressor genes. In addition, we report the cloning of the cDNAs for genes related toS. pombe rad17, rad9, rad1,andhus1from mouse,Caenorhabditis elegans,andDrosophila melanogaster.These includeRad17andRad9fromD. melanogaster,hpr-17 and hpr-1 fromC. elegans,and RAD1 and HUS1 from mouse. The identification of homologs of theS. pomberad checkpoint genes from mammals, arthropods, and nematodes indicates that this cell cycle checkpoint pathway is conserved throughout eukaryotes.     

Site-specific recombination in Escherichia coli between the att sites of plasmid pSE211 from Saccharopolyspora erythraea

Summary pSE211 fromSaccharopolyspora erythraea integrates site-specifically into the chromosome through conservative recombination betweenattP andattB, the plasmid and chromosomal attachment sites. Integration depends on the presence ofint, an open reading frame (ORF) that lies adjacent toattP and encodes the putative integrase. Immediately upstream ofint liesxis (formerly calledorf2) which encodes a basic protein that is thought to exhibit DNA binding.xis andint were cloned in various combinations in pUC18 and expressed constitutively inEscherichia coli from thelac promoter.attP andattB were cloned inStreptomyces orE. coli plasmids containing kanamycin resistance (Km^R) or chloramphenicol resistance (Cm^R) markers. Stable Km^R Cm^R cointegrates formed byattP ×attB orattP ×attP recombination (integration) were obtained inE. coli hosts that expressedint. Co-integrates were not found in hosts expressingint+xis. Excision (intraplasmidatt site recombination) was examined by constructing plasmids carryingattL andattR or twoattP sites separating Cm^R from Km^R and by following segregation of the markers in various hosts. BothattL ×attR andattP ×attP excision depended on bothxis andint inE. coli. pSE211att site integration and excision were not affected by a deletion inhimA, the gene encoding a subunit of integration host factor.     

The structure ofHLA-B35 suggests that it is derived fromHLA-Bw58 by two genetic mechanisms

The primary structure ofHLA-B51 andHLA-Bw52 suggested thatHLA-B51 was derived fromHLA-Bw52 by the combination of a genetic exchange withHLA-B8 and a point mutation. To investigate the evolution of theHLA-B5 cross reactive group, theHLA-B35 gene was cloned and the primary structure was determined.HLA-B35 is identical toHLA-Bw58 except in the α₁ domain. The α₁ domain ofHLA-B35 except Bw4/Bw6-associated amino acids is identical to that ofHLA-B51 ^*, which was suspected to be an intermediate gene betweenHLA-B51 andHLA-Bw52. These data suggest thatHLA-B35 has evolved fromHLA-Bw58 in two steps; an in vivo replacement of the α₁ domain withHLA-B51 and genetic exchange with one of theHLA-Bw6 genes. These three genes andHLA-Bw58 are postulated to share a common ancestor.     

Analyses oftraA, int-Tn,andxis-TnMutations in the Conjugative Transposon Tn916inEnterococcus faecalis

The conjugative transposon Tn916moves intercellularly via an excision/insertion mechanism that involves products ofint-Tnandxis-Tn.Tn5-insertion mutations in these genes were found to be complemented in anEnterococcus faecalishost by specific coresident transposons harboring the corresponding wild-type allele. A determinant designatedtraA,partially overlapping and divergently transcribed fromxis-Tn,is thought to encode a key positively acting regulatory protein needed for expression of conjugation functions. This locus was also shown to express atrans-acting product.     

TheAspergillus nidulans geneschsA andchsD encode chitin synthases which have redundant functions in conidia formation

We previously isolated three chitin synthase genes (chsA, chsB, andchsC) fromAspergillus nidulans. In the present work, we describe the isolation and characterization of another chitin synthase gene, namedchsD, fromA. nidulans. Its deduced amino acid sequence shows 56.7% and 55.9% amino acid identity, respectively, with Cal1 ofSaccharomyces cerevisiae and Chs3 ofCandida albicans. Disruption ofchsD caused no defect in cell growth or morphology during the asexual cycle and caused no decrease in chitin content in hyphae. However, double disruption ofchsA andchsD caused a remarkable decrease in the efficiency of conidia formation, while double disruption ofchsC andchsD caused no defect. Thus it appears thatchsA andchsD serve redundant functions in conidia formation.     

Identification and fine mapping of AvrPi15, a novel avirulence gene of Magnaporthe grisea

Avirulence of Magnaporthe grisea isolate CHL346 on rice cultivar GA25 was studied with 242 ascospore progenies derived from the cross CHL346 × CHL42. Segregation analysis of the avirulence in the progeny population was in agreement with the existence of a single avirulence (Avr) gene, designated as AvrPi15. For mapping the Avr gene, we developed a total of 121 microsatellite DNA markers [simple sequence repeat (SSR)], which evenly distributed in the whole-genome of M. grisea through bioinformatics analysis (BIA) using the publicly available sequence. Linkage analysis of the AvrPi15 gene with these SSR markers showed that six markers on chromosome 6, MS6-1, MS6-2, MS6-3, MS6-7, MS6-8 and MS6-10, were linked to the AvrPi15 locus. To further define the chromosomal location of the AvrPi15 locus, two additional markers, MS6-17 and STS6-6, which were developed based on the sequences of telomeric region 11 (TEL11), were subjected to linkage analysis. The results showed that MS6-17 and STS6-6 were associated with the locus by 3.3 and 0.8 cM, respectively. To finely map the Avr gene, two additional candidate avirulence gene (CAG) markers, CAG6-1 and CAG6-2, were developed based on the gene annotation of the sequence of TEL 11. Linkage analysis of the Avr gene with these two markers revealed that both of them completely cosegregated with the AvrPi15 locus. Finally, this locus was physically mapped into ∼ 7.2-kb interval of the TEL11 by BIA using these sequence-ready markers. This is the key step toward positional cloning of the AvrPi15 gene.     

Discriminant haplotypes of avirulence genes of Phytophthora sojae lead to a molecular assay to predict phenotypes

The soybean–Phytophthora sojae interaction operates on a gene-for-gene relationship, where the product of a resistance gene (Rps) in the host recognizes that of an avirulence gene (Avr) in the pathogen to generate an incompatible reaction. To exploit this form of resistance, one must match with precision the appropriate Rps gene with the corresponding Avr gene. Currently, this association is evaluated by phenotyping assays that are labour-intensive and often imprecise. To circumvent this limitation, we sought to develop a molecular assay that would reveal the avirulence allele of the seven main Avr genes (Avr1a, Avr1b, Avr1c, Avr1d, Avr1k, Avr3a, and Avr6) in order to diagnose with precision the pathotypes of P. sojae isolates. For this purpose, we analysed the genomic regions of these Avr genes in 31 recently sequenced isolates with different virulence profiles and identified discriminant mutations between avirulence and virulence alleles. Specific primers were designed to generate amplicons of a distinct size, and polymerase chain reaction conditions were optimized in a final assay of two parallel runs. When tested on the 31 isolates of known virulence, the assay accurately revealed all avirulence alleles. The test was further assessed and compared to a phenotyping assay on 25 isolates of unknown virulence. The two assays matched in 97% (170/175) of the interactions studied. Interestingly, the sole cases of discrepancy were obtained with Avr3a, which suggests a possible imperfect interaction with Rps3a. This molecular assay offers a powerful and reliable tool to exploit and study with greater precision soybean resistance against P. sojae.     

NUT1, a major nitrogen regulatory gene inMagnaporthe grisea,is dispensable for pathogenicity

NUT1, a gene homologous to the major nitrogen regulatory genesnit-2 ofNeurospora crassa andareA ofAspergillus nidulans, was isolated from the rice blast fungus,Magnaporthe grisea. NUT1 encodes a protein of 956 amino acid residues and, likenit-2 andareA, has a single putative zinc finger DNA-binding domain. Functional equivalence ofNUT1 toareA was demonstrated by introducing theNUT1 gene by DNA-mediated transformation into anareA loss-of-function mutant ofA. nidulans. The introducedNUT1 gene fully complemented theareA null mutation, restoring to the mutant the ability to utilize a variety of nitrogen sources. In addition, the sensitivity ofAspergillus NUT1 transformants to ammonium repression of extracellular protease activity was comparable to that of wild-typeA. nidulans. Thus,NUT1 andareA encode functionally equivalent gene products that activate expression of nitrogen-regulated genes. A one-step gene disruption strategy was used to generatenutl ^? mutants ofM. grisea by transforming a rice-infecting strain with a disruption vector in which a gene for hygromycin B phosphotransferase (Hyg) replaced the zinc-finger DNA-binding motif ofNUT1. Of 31 hygromycin B (hyg B)-resistant transformants shown by Southern hybridization to contain a disruptedNUT1 gene (nut1::Hyg), 26 resulted from single-copy replacement events at theNUT1 locus. Althoughnut1 ^? transformants ofM. grisea failed to grown on a variety of nitrogen sources, glutamate, proline and alanine could still be utilized. This contrasts withA. nidulans where disruption of the zinc-finger region ofareA prevents utilization of nitrogen sources other than ammonium and glutamine. The role ofNUT1 and regulation of nitrogen metabolism in the disease process was evaluated by pathogenicity assays. The infection efficiency ofnut1 ^? transformants on susceptible rice plants was similar to that of the parental strain, although lesions were reduced in size. These studies demonstrate that theM. grisea NUT1 gene activates expression of nitrogen-regulated genes but is dispensable for pathogenicity.     

Cloning and expression analysis of a predicted toxin gene fromPhotorhabdus sp. HB78

The genome of the insect pathogenPhotorhabdus luminescens TT01 strain contains multiple genes predicted to encode toxins. One of these,plu0840, has 55% sequence identity with an enterotoxin fromAeromonas hydrophila. In order to further investigate this gene, we successfully cloned the completeplu0840 fromPhotorhabdus sp. HB78 and expressed it as a GST-Plu0840 fusion protein inEscherichia coli BL21 (DE3) using pGEX-4T-1 as a vector. Most of GST-Plu0840 was insoluble and sequestered into inclusion bodies. The inclusion bodies were harvested and dissolved. The resultant protein was cleaved and purified from the GST-tag. Oral bioassay showed that Plu0840 inhibited growth ofSpodoptera litura andSpodoptera exigua larvae.