Molecular heterogeneity of XY sex reversal in horses.

Male-to-female 64,XY sex reversal is a frequently reported chromosome abnormality in horses. Despite this, the molecular causes of the condition are as yet poorly understood. This is partially because only limited molecular information is available for the horse Y chromosome (ECAY). Here, we used the recently developed ECAY map and carried out the first comprehensive study of the Y chromosome in XY mares (n=18). The integrity of the ECAY in XY females was studied by FISH and PCR using markers evenly distributed along the euchromatic region. The results showed that the XY sex reversal condition in horses has two molecularly distinct forms: (i) a Y-linked form that is characterized by Y chromosome deletions and (ii) a non-Y-linked form where the Y chromosome of affected females is molecularly the same as in normal males. Further analysis of the Y-linked form (13 cases) showed that the condition is molecularly heterogeneous: the smallest deletions spanned about 21 kb, while the largest involved the entire euchromatic region. Regardless of the size, all deletions included the SRY gene. We show that the deletions were likely caused by inter-chromatid recombination events between repeated sequences in ECAY. Further, we hypothesize that the occurrence of SRY-negative XY females in some species (horse, human) but not in others (pig, dog) is because of differences in the organization of the Y chromosome. Finally, in contrast to the Y-linked SRY-negative form of equine XY sex reversal, the molecular causes of SRY-positive XY mares (5 cases) remain as yet undefined.

A fatty acid desaturase modulates the activation of defense signaling pathways in plants.

Salicylic acid (SA) plays an important role in activating various plant defense responses, including expression of the pathogenesis-related (PR) genes and systemic acquired resistance. A critical positive regulator of the SA signaling pathway in Arabidopsis is encoded by the NPR1 gene. However, there is growing evidence that NPR1-independent pathways can also activate PR expression and disease resistance. To elucidate the components associated with NPR1-independent defense signaling, we isolated a suppressor of the npr1-5 allele, designated ssi2. The recessive ssi2 mutation confers constitutive PR gene expression, spontaneous lesion formation, and enhanced resistance to Peronospora parasitica. In contrast, a subset of defense responses regulated by the jasmonic acid (JA) signaling pathway, including expression of the defensin gene PDF1.2 and resistance to Botrytis cinerea, is impaired in ssi2 plants. With the use of a map-based approach, the SSI2 gene was cloned and shown to encode a stearoyl-ACP desaturase (S-ACP DES). S-ACP DES is an archetypical member of a family of soluble fatty acid (FA) desaturases; these enzymes play an important role in regulating the overall level of desaturated FAs in the cell. The activity of mutant S-ACP DES enzyme was reduced 10-fold, resulting in elevation of the 18:0 FA content in ssi2 plants. Because reduced S-ACP DES activity leads to the induction of certain defense responses and the inhibition of others, we propose that a FA-derived signal modulates crosstalk between different defense signaling pathways.

Environmentally sensitive, SA-dependent defense responses in the cpr22 mutant of Arabidopsis.

To investigate the signaling pathways through which defense responses are activated following pathogen infection, we have isolated and characterized the cpr22 mutant. This plant carries a semidominant, conditional lethal mutation that confers constitutive expression of the pathogenesis-related (PR) genes PR-1, PR-2, PR-5 and the defensin gene PDF1.2. cpr22 plants also display spontaneous lesion formation, elevated levels of salicylic acid (SA) and heightened resistance to Peronospora parasitica Emco5. The cpr22 locus was mapped to chromosome 2, approximately 2 cM telomeric to the AthB102 marker. By analyzing the progeny of crosses between cpr22 plants and either NahG transgenic plants or npr1 mutants, all of the cpr22-associated phenotypes except PDF1.2 expression were found to be SA dependent. However, the SA signal transducer NPR1 was required only for constitutive PR-1 expression. A cross between cpr22 and ndr1-1 mutants revealed that enhanced resistance to P. parasitica is mediated by an NDR1-dependent pathway, while the other cpr22-induced defenses are not. Crosses between either coi1-1 or etr1-1 mutants further demonstrated that constitutive PDF1.2 expression is mediated by a JA- and ethylene-dependent pathway. Based on these results, the cpr22 mutation appears to induce its associated phenotypes by activating NPR1-dependent and NPR1-independent branches of the SA pathway, as well as an ethylene/JA signaling pathway. Interestingly, the SA-dependent phenotypes, but not the SA-independent phenotypes, are suppressed when cpr22 mutants are grown under high humidity.

A recessive mutation in the Arabidopsis SSI2 gene confers SA- and NPR1-independent expression of PR genes and resistance against bacterial and oomycete pathogens.

The Arabidopsis thaliana NPR1 gene is required for salicylic acid (SA)-induced expression of pathogenesis-related (PR) genes and systemic acquired resistance. However, loss-of-function mutations in NPR1 do not confer complete loss of PR gene expression or disease resistance. Thus these responses also can be activated via an NPR1-independent pathway that currently remain to be elucidated. The ssi2-1 mutant, identified in a genetic screen for suppressors of npr1-5, affects signaling through the NPR1-independent defense pathway(s). In comparison with the wild-type (SSI2 NPR1) plants and the npr1-5 mutant (SSI2 npr1-5), the ssi2-1 npr1-5 double mutant and the ssi2-1 NPR1 single mutant constitutively express PR genes [PR-1, BGL2 (PR-2) and PR-5]; accumulate elevated levels of SA; spontaneously develop lesions; and possess enhanced resistance to a virulent strain of Peronospora parasitica. The ssi2-1 mutation also confers enhanced resistance to Pseudomonas syringae pv. tomato (Pst); however, this is accomplished primarily via an NPR1-dependent pathway. Analysis of ssi2-1 NPR1 nahG and ssi2-1 npr1-5 nahG plants revealed that elevated SA levels were not essential for the ssi2-1-conferred phenotypes. However, expression of the nahG transgene did reduce the intensity of some ssi2-1-conferred phenotypes, including PR-1 expression, and disease resistance. Based on these results, SSI2 or an SSI2-generated signal appears to modulate signaling of an SA-dependent, NPR1-independent defense pathway, or an SA- and NPR1-independent defense pathway.

Nitric oxide and salicylic acid signaling in plant defense.

Salicylic acid (SA) plays a critical signaling role in the activation of plant defense responses after pathogen attack. We have identified several potential components of the SA signaling pathway, including (i) the H(2)O(2)-scavenging enzymes catalase and ascorbate peroxidase, (ii) a high affinity SA-binding protein (SABP2), (iii) a SA-inducible protein kinase (SIPK), (iv) NPR1, an ankyrin repeat-containing protein that exhibits limited homology to IkappaBalpha and is required for SA signaling, and (v) members of the TGA/OBF family of bZIP transcription factors. These bZIP factors physically interact with NPR1 and bind the SA-responsive element in promoters of several defense genes, such as the pathogenesis-related 1 gene (PR-1). Recent studies have demonstrated that nitric oxide (NO) is another signal that activates defense responses after pathogen attack. NO has been shown to play a critical role in the activation of innate immune and inflammatory responses in animals. Increases in NO synthase (NOS)-like activity occurred in resistant but not susceptible tobacco after infection with tobacco mosaic virus. Here we demonstrate that this increase in activity participates in PR-1 gene induction. Two signaling molecules, cGMP and cyclic ADP ribose (cADPR), which function downstream of NO in animals, also appear to mediate plant defense gene activation (e.g., PR-1). Additionally, NO may activate PR-1 expression via an NO-dependent, cADPR-independent pathway. Several targets of NO in animals, including guanylate cyclase, aconitase, and mitogen-activated protein kinases (e.g., SIPK), are also modulated by NO in plants. Thus, at least portions of NO signaling pathways appear to be shared between plants and animals.

Members of the Arabidopsis HRT/RPP8 family of resistance genes confer resistance to both viral and oomycete pathogens.

Turnip crinkle virus (TCV) inoculation onto TCV-resistant Arabidopsis leads to a hypersensitive response (HR) controlled by the dominant gene HRT. HRT is a member of the class of resistance (R) genes that contain a leucine zipper, a nucleotide binding site, and leucine-rich repeats. The chromosomal position of HRT and its homology to resistance gene RPP8 and two RPP8 homologs indicate that unequal crossing over and gene conversion may have contributed to HRT evolution. RPP8 confers resistance to an oomycete pathogen, Peronospora parasitica. Despite very strong similarities within the HRT/RPP8 family, HRT and RPP8 are specific for the respective pathogens they detect. Hence, the HRT/RPP8 family provides molecular evidence that sequence changes between closely related members of multigene families can generate novel specificities for radically different pathogens. Transgenic plants expressing HRT developed an HR but generally remained susceptible to TCV because of a second gene, RRT, that regulates resistance to TCV. However, several transgenic plants that overexpressed HRT produced micro-HRs or no HR when inoculated with TCV and were resistant to infection. Expression of the TCV coat protein gene in seedlings containing HRT resulted in massive necrosis and death, indicating that the avirulence factor detected by the HRT-encoded protein is the TCV coat protein.

Resistance to turnip crinkle virus in Arabidopsis is regulated by two host genes and is salicylic acid dependent but NPR1, ethylene, and jasmonate independent.

Inoculation of turnip crinkle virus (TCV) on the resistant Arabidopsis ecotype Dijon (Di-17) results in the development of a hypersensitive response (HR) on the inoculated leaves. To assess the role of the recently cloned HRT gene in conferring resistance, we monitored both HR and resistance (lack of viral spread to systemic tissues) in the progeny of a cross between resistant Di-17 and susceptible Columbia plants. As expected, HR development segregated as a dominant trait that corresponded with the presence of HRT. However, all of the F(1) plants and three-fourths of HR(+) F(2) plants were susceptible to the virus. These results suggest the presence of a second gene, termed RRT, that regulates resistance to TCV. The allele present in Di-17 appears to be recessive to the allele or alleles present in TCV-susceptible ecotypes. We also demonstrate that HR formation and TCV resistance are dependent on salicylic acid but not on ethylene or jasmonic acid. Furthermore, these phenomena are unaffected by mutations in NPR1. Thus, TCV resistance requires a yet undefined salicylic acid-dependent, NPR1-independent signaling pathway.

The Arabidopsis ssi1 mutation restores pathogenesis-related gene expression in npr1 plants and renders defensin gene expression salicylic acid dependent.

The Arabidopsis NPR1 gene was previously shown to be required for the salicylic acid (SA)- and benzothiadiazole (BTH)-induced expression of pathogenesis-related (PR) genes and systemic acquired resistance. The dominant ssi1 (for suppressor of SA insensitivity) mutation characterized in this study defines a new component of the SA signal transduction pathway that bypasses the requirement of NPR1 for expression of the PR genes and disease resistance. The ssi1 mutation caused PR (PR-1, BGL2 [PR-2], and PR-5) genes to be constitutively expressed and restored resistance to an avirulent strain of Pseudomonas syringae pv tomato in npr1-5 (previously called sai1) mutant plants. In addition, ssi1 plants were small, spontaneously developed hypersensitive response-like lesions, accumulated elevated levels of SA, and constitutively expressed the antimicrobial defensin gene PDF1.2. The phenotypes of the ssi1 mutant are SA dependent. When SA accumulation was prevented in ssi1 npr1-5 plants by expressing the SA-degrading salicylate hydroxylase (nahG) gene, all of the phenotypes associated with the ssi1 mutation were suppressed. However, lesion formation and expression of the PR genes were restored in these plants by the application of BTH. Interestingly, expression of PDF1.2, which previously has been shown to be SA independent but jasmonic acid and ethylene dependent, was also suppressed in ssi1 npr1-5 plants by the nahG gene. Furthermore, exogenous application of BTH restored PDF1.2 expression in these plants. Our results suggest that SSI1 may function as a switch modulating cross-talk between the SA- and jasmonic acid/ethylene-mediated defense signal transduction pathways.

Production and secretion of biologically active human epidermal growth factor in Yarrowia lipolytica.

The gene encoding human epidermal growth factor (hEGF) was expressed as a fusion protein with the leader peptide and pro I region of alkaline extracellular protease in the yeast Yarrowia lipolytica. hEGF was purified from culture supernatant by reverse-phase chromatography and analysed by Western-blot hybridisations. The biologically active hEGF in the purified sample was assayed using the radioreceptor assay and estimated to be 100 microg/l. However, the level of expression was found to be substantially low compared to the levels of homologous protein, alkaline extracellular protease (AEP), possibly due to degradation by secreted acid protease(s). A novel and sensitive bioassay was developed to determine the biological activity of hEGF produced at low levels and is based on the effect produced by hEGF in the regenerating tails of the wall lizard. Intramuscular injections of culture supernatant from the recombinant yeast and the standard hEGF led to a drastic reduction in tail regeneration confirming the biological activity of the recombinant hEGF.

Organisation and molecular analysis of repeated DNA sequences in the rice blast fungus Magnaporthe grisea.

The distribution of a previously described repeated DNA sequence present as a 1.3-kb PstI fragment in the genome of the rice blast fungus Magnaporthe grisea was analysed by carrying out DNA fingerprint analysis of 36 isolates including rice, non-rice and laboratory strains. The analysis of various higher-molecular-weight PstI fragments with homology to the 1.3-kb repeat revealed that these may arise predominantly from transposon insertions or point mutations. Analysis of a 5.1-kb derivative revealed both a point mutation at a PstI site and an insertion of a putative transposable element which caused an increase in molecular weight from 1.3 to 5.1 kb. Another repeat element of 1.4 kb was identified and found to exist in association with the 1.3-kb repeat. Both 1.3- and 1.4-kb elements were found to be parts of MGR583 (Hamer et al. 1989), a LINE-like element. These elements were present in a high copy number in all the rice and a majority of non-rice pathogens indicating that MGR583 is not a host-specific sequence as reported earlier. Our results suggest that repeated DNA elements in M. grisea have amplified independently of one another and further indicate that different isolates of M. grisea may have evolved from several distinct lines of origin.

Analysis of host-induced response in the rice blast fungus Magnaporthe grisea using two-dimensional polyacrylamide gel electrophoresis.

Two-dimensional (2-D) polyacrylamide gel electrophoresis of proteins was used to study the response of the rice blast fungus to extracts prepared from resistant and susceptible rice cultivars. A protein of molecular mass 31 kDa was induced by a susceptible host extract, while the fungus exposed to extract from the resistant cultivar and the untreated samples did not show the presence of this protein. Levels of this 31 kDa protein increased 30-fold, 72 h after treatment with plant extracts, with the concomitant appearance of at least sixteen other novel proteins. Fungus treated with extracts of resistant host or the untreated samples did not show any of these proteins while the proteins specific to different growth stages appeared as expected. Analysis of the extracellular samples showed induction of a 17 kDa protein after 72 h in the culture treated with susceptible host extract. Since the resistant host extract does not cause induction of any protein it is likely that the proteins induced in response to the susceptible host are expressed during the disease process and/or its establishment. Our study demonstrates usefulness of 2-D analysis in understanding host-pathogen interactions.

Mg-SINE: a short interspersed nuclear element from the rice blast fungus, Magnaporthe grisea.

A short interspersed nuclear element, Mg-SINE, was isolated and characterized from the genome of the rice blast fungus, Magnaporthe grisea. Mg-SINE was isolated as an insertion element within Pot2, an inverted-repeat transposon from M. grisea and shows typical features of a mammalian SINE. Mg-SINE is present as a 0.47-kb interspersed sequence at approximately 100 copies per haploid genome in both rice and non-rice isolates of M. grisea, indicating a common evolutionary origin. Secondary structure analysis of Mg-SINE revealed a tRNA-related region at the 5' end which folds into a cloverleaf structure. Genomic fusions resulting in chimeric Mg-SINEs (Ch-SINEs) composed of a sequence homologous to Mg-SINE at the 3' end and an unrelated sequence at its 5' end were also isolated, indicating that this and other DNA rearrangements mediated by these elements may have a major effect on the genomic architecture of this fungus.

Pot2, an inverted repeat transposon from the rice blast fungus Magnaporthe grisea.

We report the cloning and characterisation of Pot2, a putative transposable element from Magnaporthe grisea. The element is 1857 bp in size, has 43-bp perfect terminal inverted repeats (TIRs) and 16-bp direct repeats within the TIRs. A large open reading frame, potentially coding for a transposase-like protein, was identified. This putative protein coding region showed extensive identity to that of Fot1, a transposable element from another phytopathogenic fungus, Fusarium oxysporum. Pot2, like the transposable elements Tc1 and Mariner of Caenorhabditis elegans and Drosophila, respectively, duplicates the dinucleotide TA at the target insertion site. Sequence analysis of DNA flanking 12 Pot2 elements revealed similarity to the consensus insertion sequence of Tc1. Pot2 is present at a copy number of approximately 100 per haploid genome and represents one of the major repetitive DNAs shared by both rice and non-rice pathogens of M. grisea.