Evolution of a multi-step phosphorelay signal transduction system in Ensifer: recruitment of the sigma factor RpoN and a novel enhancer-binding protein triggers acid-activated gene expression.

Most Ensifer strains are comparatively acid sensitive, compromising their persistence in low pH soils. In the acid-tolerant strain Ensifer medicae WSM419, the acid-activated expression of lpiA is essential for enhancing survival in lethal acidic conditions. Here we characterise a multi-step phosphorelay signal transduction pathway consisting of TcsA, TcrA, FsrR, RpoN and its cognate enhancer-binding protein EbpA, which is required for the induction of lpiA and the downstream acvB gene. The fsrR, tcrA, tcsA and rpoN genes were constitutively expressed, whereas lpiA and acvB were strongly acid-induced. RACE mapping revealed that lpiA/acvB were co-transcribed as an operon from an RpoN promoter. In most Ensifer species, lpiA/acvB is located on the chromosome and the sequence upstream of lpiA lacks an RpoN-binding site. Nearly all Ensifer meliloti strains completely lack ebpA, tcrA, tcsA and fsrR regulatory loci. In contrast, E. medicae strains have lpiA/acvB and ebpA/tcrA/tcsA/fsrR co-located on the pSymA megaplasmid, with lpiA/acvB expression coupled to an RpoN promoter. Here we provide a model for the expression of lpiA/acvB in E. medicae. This unique acid-activated regulatory system provides insights into an evolutionary process which may assist the adaptation of E. medicae to acidic environmental niches.

Molecular Mapping of Stripe Rust Resistance Gene Yr76 in Winter Club Wheat Cultivar Tyee.

Tyee, one of the wheat cultivars used to differentiate races of Puccinia striiformis f. sp. tritici in the United States, was identified to have a single gene for all-stage resistance, tentatively named YrTye. To map the gene, Tyee was crossed with 'Avocet Susceptible' (AvS). Genetic analysis of the F1, F2, F2:3, and BC1 progenies confirmed a single dominant gene for resistance to race PSTv-37 that is avirulent to YrTye. A mapping population of 135 F2 plants was phenotyped with PSTv-37 and the derived F2:3 lines were tested with races PSTv-37, PSTv-40, and PSTv-79. The F2 mapping population was genotyped with simple sequence repeat (SSR) markers. A genetic map comprising 13 SSR markers located YrTye in chromosome 3AS flanked distally by SSR marker wmc11 and proximally by wmc532 at 2.6 and 3.4 cM, respectively. Amplification of Chinese Spring 3A deletion lines placed the gene in the distal bin 3AS4-0.45 to 1.00. Because YrTye is different from all formally named Yr genes in chromosomal location, we permanently name the gene Yr76. A near-isogenic line of spring common wheat was developed and selected by testing F3 lines derived from a AvS*4/Tyee cross with Tyee-avirulent and virulent races and the flanking markers. The specific SSR alleles flanking Yr76 were validated using cultivars and breeding lines with and without the gene, and showed high polymorphisms. The specificity of Yr76 is useful in differentiating P. striiformis f. sp. tritici races, and its tightly linked markers will be useful in developing resistant cultivars when combining the gene with other genes for resistance to stripe rust.

Molecular Mapping of YrSP and Its Relationship with Other Genes for Stripe Rust Resistance in Wheat Chromosome 2BL.

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is an important disease of wheat worldwide. Resistance is the best way to control the disease. YrSP, a gene originally from 'Spaldings Prolific' wheat and providing resistance to a broad spectrum of races, is used for differentiating P. striiformis f. sp. tritici races but its chromosomal location is not clear. To map YrSP, a near-isogenic line (AvSYrSPNIL) was backcrossed to the recurrent parent, Avocet S. Genetic analysis of the BC7F1, BC8, BC7F2, and BC7F3 progenies confirmed a single dominant gene for resistance. In total, 182 BC7F2 plants and their derived BC7F3 lines were phenotyped with an avirulent P. striiformis f. sp. tritici race and genotyped with simple-sequence repeat (SSR), single-nucleotide polymorphism (SNP), and sequence-tagged site (STS) markers. A linkage map was constructed with 3 SSR, 17 SNP, and 3 STS markers covering 23.3 centimorgans (cM). Markers IWA638 and dp269 were 0.6 cM proximal and 1.5 cM distal, respectively, to YrSP. The gene was mapped in chromosome bin 2BL-C-0.5, physically within the proximal 50% of the chromosome 2BL arm. Allelism tests based on F2 phenotypes indicated that YrSP is closely linked to but not allelic with genes Yr5, Yr7, Yr43, Yr44, and Yr53. Infection type data from tests with 10 historical and currently predominant P. striiformis f. sp. tritici races in the United States also demonstrated differences in specificity between YrSP and the other genes. The specificity of YrSP is useful in differentiating P. striiformis f. sp. tritici races and studying the plant-pathogen interactions, and the information of chromosomal location of the gene and its tightly linked markers should be useful in developing resistant cultivars when combined with other genes for resistance to stripe rust.

Barberry as Alternate Host Is Important for Puccinia graminis f. sp. tritici But Not for Puccinia striiformis f. sp. tritici in the U.S. Pacific Northwest.

Common barberry (Berberis vulgaris) is the alternate host of the wheat stem rust pathogen, Puccinia graminis f. sp. tritici, under natural conditions in the U.S. Pacific Northwest. Barberry was recently shown to be infected by basidiospores of the wheat stripe rust pathogen, Puccinia striiformis f. sp. tritici, under controlled conditions, but it is unclear if barberry plays any role in stripe rust epidemics under natural conditions. Aecial samples of Puccinia spp. collected from barberry plants in the Pacific Northwest from 2010 to 2013 were characterized to species by inoculation on wheat plants under controlled conditions and by molecular markers and sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. Inoculation of wheat plants with bulked aecia-bearing barberry samples resulted in most P. graminis f. sp. tritici uredia and some P. striiformis f. sp. tritici uredinia. Virulence tests demonstrated that the P. graminis f. sp. tritici isolates were sexually produced, whereas the P. striiformis f. sp. tritici isolates were clonal based on both virulence and simple sequence repeat marker tests, indicating urediniospores from wheat fields landing on barberry leaves as the possible source of P. striiformis f. sp. tritici inoculum. A method for simultaneously testing individual aecia for identifying of P. graminis f. sp. tritici and P. striiformis f. sp. tritici by pathogenicity and ITS markers. Using the method together with ITS sequencing, tested individual aecia were mostly P. graminis f. sp. tritici and occasionally some other formae speciales of P. graminis, but not P. striiformis. The results imply that barberry is essential for stem rust epidemics, but not for stripe rust under the natural conditions in the U.S. Pacific Northwest.

Virulence Characterization of International Collections of the Wheat Stripe Rust Pathogen, Puccinia striiformis f. sp. tritici.

Wheat stripe rust (yellow rust [Yr]), caused by Puccinia striiformis f. sp. tritici, is an economically important disease of wheat worldwide. Virulence information on P. striiformis f. sp. tritici populations is important to implement effective disease control with resistant cultivars. In total, 235 P. striiformis f. sp. tritici isolates from Algeria, Australia, Canada, Chile, China, Hungary, Kenya, Nepal, Pakistan, Russia, Spain, Turkey, and Uzbekistan were tested on 20 single Yr-gene lines and the 20 wheat genotypes that are used to differentiate P. striiformis f. sp. tritici races in the United States. The 235 isolates were identified as 129 virulence patterns on the single-gene lines and 169 virulence patterns on the U.S. differentials. Virulences to YrA, Yr2, Yr6, Yr7, Yr8, Yr9, Yr17, Yr25, YrUkn, Yr28, Yr31, YrExp2, Lemhi (Yr21), Paha (YrPa1, YrPa2, YrPa3), Druchamp (Yr3a, YrD, YrDru), Produra (YrPr1, YrPr2), Stephens (Yr3a, YrS, YrSte), Lee (Yr7, Yr22, Yr23), Fielder (Yr6, Yr20), Tyee (YrTye), Tres (YrTr1, YrTr2), Express (YrExp1, YrExp2), Clement (Yr9, YrCle), and Compair (Yr8, Yr19) were detected in all countries. At least 80% of the isolates were virulent on YrA, Yr2, Yr6, Yr7, Yr8, Yr17, YrUkn, Yr31, YrExp2, Yr21, Stephens (Yr3a, YrS, YrSte), Lee (Yr7, Yr22, Yr23), and Fielder (Yr6, Yr20). Virulences to Yr1, Yr9, Yr25, Yr27, Yr28, Heines VII (Yr2, YrHVII), Paha (YrPa1, YrPa2, YrPa3), Druchamp (Yr3a, YrD, YrDru), Produra (YrPr1, YrPr2), Yamhill (Yr2, Yr4a, YrYam), Tyee (YrTye), Tres (YrTr1, YrTr2), Hyak (Yr17, YrTye), Express (YrExp1, YrExp2), Clement (Yr9, YrCle), and Compair (Yr8, Yr19) were moderately frequent (>20 to <80%). Virulence to Yr10, Yr24, Yr32, YrSP, and Moro (Yr10, YrMor) was low (≤20%). Virulence to Moro was absent in Algeria, Australia, Canada, Kenya, Russia, Spain, Turkey, and China, but 5% of the Chinese isolates were virulent to Yr10. None of the isolates from Algeria, Canada, China, Kenya, Russia, and Spain was virulent to Yr24; none of the isolates from Algeria, Australia, Canada, Nepal, Russia, and Spain was virulent to Yr32; none of the isolates from Australia, Canada, Chile, Hungary, Kenya, Kenya, Nepal, Pakistan, Russia, and Spain was virulent to YrSP; and none of the isolates from any country was virulent to Yr5 and Yr15. Although the frequencies of virulence factors were different, most of the P. striiformis f. sp. tritici isolates from these countries shared common virulence factors. The virulences and their frequencies and distributions should be useful in breeding stripe-rust-resistant wheat cultivars and understanding the pathogen migration and evolution.

Evidence of genetic recombination in wheat yellow rust populations of a Chinese oversummering area.

Wheat yellow rust (Puccinia striiformis f.sp. tritici) (PST) has been described as a strongly clonal species in both European and Australian populations, with very limited molecular diversity but rapidly evolving virulences. Contrastingly, marked genetic diversity has been reported in Chinese PST populations. To test whether such variability could originate from oversummering areas, we assessed the diversity of virulence and molecular markers (AFLP and SSR) using 412 PST isolates from the highlands of Tianshui county in Gansu province. Very marked phenotypic and genotypic diversity (38% and 89%, respectively) was found. No genetic structure dependent on the sites sampled (Fst=0.004) or altitude distribution (Fst=0.0098) was detected, indicating important gene flow at the county scale. This study also revealed genetic recombination between molecular markers and thus strongly suggests the existence of a sexual or parasexual cycle in PST in Tianshui county. The observations of higher rates of sexual spore production in genotypes originating from Tianshui are the very first elements suggestive of the existence of a sexual cycle in this species.

Seedling and Slow Rusting Resistance to Stripe Rust in Chinese Common Wheats.

Identification of seedling and slow stripe rust resistance genes is important for gene pyramiding, gene deployment, and developing slow-rusting wheat cultivars to control the disease. A total of 98 Chinese lines were inoculated with 26 pathotypes of Puccinia striiformis f. sp. tritici for postulation of stripe rust resistance genes effective at the seedling stage. A total of 135 wheat lines were planted at two locations to characterize their slow rusting responses to stripe rust in the 2003-2004 and 2004-2005 cropping seasons. Genes Yr2, Yr3a, Yr4a, Yr6, Yr7, Yr9, Yr26, Yr27, and YrSD, either singly or in combinations, were postulated in 72 lines, whereas known resistance genes were not identified in the other 26 accessions. The resistance genes Yr9 and Yr26 were found in 42 and 19 accessions, respectively. Yr3a and Yr4a were detected in two lines, and four lines may contain Yr6. Three lines were postulated to possess YrSD, one carried Yr27, and one may possess Yr7. Thirty-three lines showed slow stripe rusting resistance at two locations in both seasons.

Structural features of an antigen required for cellular interactions and for T cell activation in a MHC-restricted response.

The protein Ag, tobacco mosaic virus protein, (TMVP) and its tryptic peptide number 8 (residues 93-112 of the protein) exhibit cross-reactivity on the T cell level in some strains of mice (e.g., C3H.SW, C57BL/10); these strains are termed cross-reactive (CR). In other strains such as A/J or B10.BR, no cross-reactivity is exhibited; these strains are termed non-cross-reactive (NCR). Genetic experiments indicated that the cross-reactivity is dominant and that it is mapped to the I-A or I-E region of the MHC, with cross-reactivity exhibited by the I-Ab haplotype but not by I-Ak or I-Ek. Cell reconstitution experiments have indicated that the non-cross-reactivity is associated with the inability of the NCR APC to present Ag. Analysis of the area(s) on peptide 8 which serve(s) as epitope revealed that both strains recognize an overlapping area consisting of 11 amino acid residues in the middle of peptide 8 (residues 97-107), which by itself is nonstimulatory to TMVP- or peptide 8-immune T cells of the CR or the NCR strains. However, the addition of a few amino acid residues of the sequence of peptide 8 to this area converts it to a complete stimulatory epitope. Additivity experiments revealed that the CR strain contains two major T cell populations each recognizing this middle region of peptide 8 when elongated by a few amino acids N-terminally and C-terminally, respectively. In contrast, the NCR strain contains one major T cell population recognizing elongation only N-terminally. Because TMVP (but not peptide 8) requires processing before presentation to T cells, it is postulated that, during processing of TMVP, there occur alterations in the area of the proximal three or four N-terminal amino acids of the region consisting of peptide 8, destroying the only region containing the T cell epitope recognized by the NCR strain, hence TMVP and peptide 8 do not exhibit cross-reactivity in this strain. The same alterations of TMVP still leave intact an epitope consisting of amino acid residues C-terminal to the altered area which is recognized by the CR strain, hence the cross-reactivity exhibited by this strain. The results suggest that the difference in cross-reactivity on the T cell level between TMVP and peptide 8 exhibited by the strains may be due to differences in the orientation of presentation and the subsequent cell recognition of an epitope contained within peptide 8.(ABSTRACT TRUNCATED AT 400 WORDS)

Nature of polymorphism in HLA-A, -B, and -C molecules.

Diversity in 39 HLA-A, -B, and -C molecules is derived from 20 amino acid positions of high variability and 71 positions of low variability. Variation in the structurally homologous alpha 1 and alpha 2 domains is distinct and may correlate with partial segregation of peptide and T-cell receptor binding functions. Comparison of 15 HLA-A with 20 HLA-B molecules reveals considerable locus-specific character, due primarily to differences at polymorphic residues. The results indicate that genetic exchange between alleles of the same locus has been a more important mechanism in the generation of HLA-A, -B, and -C diversity than genetic exchange events between alleles of different loci.

Multiple genetic mechanisms have contributed to the generation of the HLA-A2/A28 family of class I MHC molecules.

The genetic events that produce diversity in class I MHC genes and proteins has been investigated by using a family of closely related HLA-A alleles. Five genes coding for HLA-A2.2Y, HLA-A2.3, and HLA-Aw68.2 have been isolated. Exon sequences are compared with the known sequences for HLA-A2.1, HLA-A2.2F, HLA-A2.4, HLA-Aw68.1, and HLA-Aw69. Pairwise comparison of the eight unique sequences shows that point mutation, reciprocal recombination, and gene conversion have all contributed significantly to the diversification of this family of alleles. These results are compared with those of other studies that have emphasized the role of gene conversion. A predominance of coding substitutions in the alpha 1 and alpha 2 domains is found, consistent with positive selection for polymorphism being a major factor in the fixation of these alleles. In the three cases examined, genes for phenotypically identical proteins gave identical nucleotide sequences, indicating that most, if not all, of the class I polymorphism is detectable by immunological methods. The apparent stability of the sequences suggests that the events generating some of the alleles occurred before the origin of modern Homo sapiens.

A transposable epitope of HLA-B7, B40 molecules.

The monoclonal antibody MB40.2 defines a novel subtype of HLA-B40 that is expressed by the Sweig cell line. This molecule, called HLA-B40, lacks an antigenic determinant that is common to HLA-B7 and the HLA-Bw60 subtype of HLA-B40. Genes encoding HLA-B40 and HLA-Bw60 have now been isolated and the amino acid sequences of these proteins compared with other HLA-B locus molecules. These results show that HLA-B40 is a unique protein which differs from HLA-Bw60 by eight amino acid substitutions. Comparison of the sequences for HLA-B40, -Bw60, and -B7 localizes the MB40.2 epitope to a cluster of three substitutions at positions 177, 178, and 180 at the end of the alpha 2 domain. Gene conversion or reciprocal recombination are postulated to have transferred this cluster of substitutions, and their associated epitope, during the evolution of HLA-B locus genes. The epitope may consist of an alpha helical segment which is exclusively found on MB40.2-positive molecules.

The primary structure of HLA-A32 suggests a region involved in formation of the Bw4/Bw6 epitopes.

All HLA-B locus molecules have either the Bw4 or Bw6 epitopes. In addition, the Bw4 epitope is found on HLA-Aw23, Aw24, and A32, and Bw6 is also found on HLA-Cw3. The structural basis for these determinants and the evolution of their distribution among products of the HLA-B locus has been a long standing puzzle. To identify residues that may be involved in these determinants, we have cloned a gene for A32 and sequenced the protein encoding exons. Comparison of the predicted protein sequence with other HLA-A,B,C sequences identified residues 79 through 83 of the alpha 1 domain as having a pattern of polymorphic substitution that correlates with the presence and absence of the Bw4 and Bw6 epitopes.

Antigenic requirements for T-cell activation: reconstitution of a functional antigen from an inactive peptide portion of an antigen conjugated to protein carriers.

The structural features of an antigenic peptide required for T-cell activation were examined by a novel approach: an active antigen was constructed from an inactive peptide portion of the original antigen by conjugating it to various proteins. An eicosapeptide, peptide 8, representing residues 103-112 of the tobacco mosaic virus protein (TMVP), was utilized as the model antigen for these studies. While peptide 8 was able to stimulate, in vitro, T-cells from peptide 8 primed mice, synthetic peptides representing various portions of peptide 8 were unable to activate these cells. Although the amino-terminal undecapeptide of peptide 8 (residues 93-103 of TMVP) was unable to activate T-cells from peptide 8 primed mice, conjugates which consisted of this undecapeptide coupled to certain proteins were capable of inducing antigen-specific proliferation of these T-cells. These results identify two structural antigenic features essential for T-cell activation: a T-cell-recognizable epitope within the amino-terminal undecapeptide of peptide 8 and a second region provided by the carboxy-terminal half of peptide 8 or by protein carriers. Potential roles for this second region include providing a site for antigen interaction with Ia molecules on the antigen-presenting cell or, alternatively, providing amino acids important in stabilizing the binding of the T-cell antigen receptor. The results suggest that the recognition of this second region exhibits only a limited specificity.

Cell activation and immunogenicity.

There is presently great interest in the production of synthetic vaccines which utilize as immunogens peptides representing portions of protein antigens, either free or conjugated to protein carriers. The use of such immunogens raises questions regarding the cells which are activated an the characteristics of the resulting immune response. Using the tobacco mosaic virus protein (TMVP) as a model antigen, immune induction by the protein and by synthetic vaccines related to this protein was investigated. Specifically we have compared immune induction by the parent protein, an unconjugated eicosapeptide representing residues 93-112 of the protein, and an immunogen consisting of the C-terminal decapeptide of the above eicosapeptide conjugated to the protein carrier KLH. The comparison led to the following conclusions: Immunization with the free eicosapeptide but not with its C-terminal decapeptide leads to the activation of T and B lymphocytes. Immunization with the free eicosapeptide or with its C-terminal decapeptide-KLH conjugate induces antibodies capable of reacting with the parent protein. The isotype composition of the antibodies induced by these immunogens is similar to that induced by immunization with the whole protein. The fine specificity of the antibodies induced by all three immunogens is similar. However, the antibody populations induced by the synthetic immunogens may be devoid of one or more clonotypes depending upon constraints imposed by cellular interaction. Antigen specific T helper cells do not seem to influence the fine specificity of antibodies induced to a given epitope. Comparison of the induction of memory responses by the three immunogens led to the conclusion that immunization with the peptide hapten conjugated to the heterologous carrier KLH does not lead to an anamnestic antibody response upon encounter with the native protein. Immunization with an immunogenic peptide representing a portion of the protein recognized by T and B lymphocytes leads to an anamnestic antibody response upon encounter with the native protein.

Immune induction by a protein antigen and by a peptide segment of the protein.

The immune induction by a protein (the tobacco mosaic virus protein-TMVP) was compared to the immune induction by the free, non-conjugated eicosa tryptic peptide fragment of the protein (tryptic peptide 8 representing residues 93-112 of the protein). The results demonstrated that like TMVP, peptide 8 was immunogenic in A/J mice. TMVP and peptide 8 do not cross react on the T cell level. However, immunization with TMVP or with peptide 8 induces antibodies which react with both TMVP and peptide 8. Characterization of the antibodies produced by both immunogens revealed that: their isotope composition is similar with IgG1 and IgG2 being the predominant isotypes; this composition indicates that both immunogens are T cell dependent antigens, the antibodies induced by TMVP and by peptide 8 are directed against the C-terminal decapeptide portion of peptide 8 (residues 103-112 of the protein), the fine specificity of these antibodies is the same. These results, and results of adoptive transfer experiments, indicate that antigen specific T cells had no effect on the expression of the fine antibody specificity. The results demonstrate the feasibility of immunizing with a portion of a protein for the purpose of inducing antibodies with the same isotype composition and specificity towards a protein epitope as those induced by immunization with the whole protein.