Isolation and characterization of a wheat IF2 homolog required for innate immunity to stripe rust

Key message

The wheat eIF2 homolog, TaIF2, is induced by the stripe rust pathogen CYR 32 at an early stage of inoculation and is related to the innate immunity resistance level in wheat.

Abstract

The initiation of translation represents a critical control point in the regulation of gene expression in all organisms. We previously identified an upregulated EST S186 (EL773056) from an SSH-cDNA library of the Shaanmai 139 strain of wheat (Triticum aestivum) infected with Puccinia striiformis (Pst). In the present work, we isolated a cDNA clone and identified it as a wheat IF2 homolog. This cDNA consisted of 1,314 nucleotides and contained an open reading frame of 795 nucleotides encoding a polypeptide of 254 amino acids. The amino acids represent a conserved domain in EF-Tu, mtIF2-II, and mtIF2-Ivc. The alignment result showed that it maybe a partial cDNA of the initiation factor 2/eukaryotic initiation factor 5B (IF2/eIF5B) superfamily gene. Paradoxically, results of a Swiss-model analysis suggesting a low QMEAN Z-score implied that it was a membrane protein. Quantitative RT-PCR studies confirmed that the wheat eIF2 (TaIF2) homolog was differentially expressed in three near-isogenic lines. Critical time points for the induction of resistance by inoculation with Pst CYR32 in YrSM139-1B + YrSM139-2D immune resistance genotype occurred at 1 and 3 dpi (days post-infection). RNAi test showed that the inoculated BSMV-IF2 leaves of Shaanmai 139 showed obvious cell death after 15 days of inoculation with CYR 32. qRT-PCR analysis of the target gene in cDNA samples isolated from BSMV-IF2-Pst, BSMV-0-Pst and Pst infected leaves confirmed that the expression of TaIF2 is suppressed by BSMV-IF2 at 3 dpi. This suggested that TaIF2/eIF5B plays an important role in the mechanism of innate immunity to stripe rust pathogen.     

TaAbc1, a Member of Abc1-Like Family Involved in Hypersensitive Response against the Stripe Rust Fungal Pathogen in Wheat

To search for genes involved in wheat (Triticum aestivum L.) defense response to the infection of stripe rust pathogen Puccinia striiformis f. sp. tritici (Pst), we identified and cloned a new wheat gene similar to the genes in the Abc1-like gene family. The new gene, designated as TaAbc1, encodes a 717-amino acid, 80.35 kD protein. The TaAbc1 protein contains two conserved domains shared by Abc1-like proteins, two trans-membrane domains at the C-terminal, and a 36-amino acid chloroplast targeting presequence at the N-terminal. Characterization of TaAbc1 expression revealed that gene expression was tissue-specific and could be up-regulated by biotic agents (e.g., stripe rust pathogen) and/or by an abiotic stress like wounding. High-fold induction was associated with the hypersensitive response (HR) triggered only by avirulent stripe rust pathotypes, suggesting that TaAbc1 is a rust-pathotype specific HR-mediator. Down-regulating TaAbc1 reduced HR but not the overall resistance level in Suwon11 to CYR23, suggesting TaAbc1 was involved in HR against stripe rust, but overall host resistance is not HR-dependent.     

西科麦2028抗条锈性的遗传分析

西科麦2028是地理远缘小麦材料的杂交后代,具有突出的抗条锈病性能。为了解西科麦2028对小麦条锈病的抗性遗传规律,以西科麦2028和铭贤169的杂交群体为研究对象,采用我国目前小麦条锈菌流行小种CYR31、CYR32、CYR33、Su11-4对供试群体进行成株期接种,分析杂交后代的抗病性及分布情况。结果表明:西科麦2028对CYR31的抗病性由3对显性基因控制;对CYR32由2对显性和1对隐性基因控制;对CYR33由1对显性基因控制;对Su11-4由1对显性和1对隐性基因控制。     

The Lr34 adult plant rust resistance gene provides seedling resistance in durum wheat without senescence

The hexaploid wheat (Triticum aestivum) adult plant resistance gene, Lr34/Yr18/Sr57/Pm38/Ltn1, provides broad‐spectrum resistance to wheat leaf rust (Lr34), stripe rust (Yr18), stem rust (Sr57) and powdery mildew (Pm38) pathogens, and has remained effective in wheat crops for many decades. The partial resistance provided by this gene is only apparent in adult plants and not effective in field‐grown seedlings. Lr34 also causes leaf tip necrosis (Ltn1) in mature adult plant leaves when grown under field conditions. This D genome‐encoded bread wheat gene was transferred to tetraploid durum wheat (T. turgidum) cultivar Stewart by transformation. Transgenic durum lines were produced with elevated gene expression levels when compared with the endogenous hexaploid gene. Unlike nontransgenic hexaploid and durum control lines, these transgenic plants showed robust seedling resistance to pathogens causing wheat leaf rust, stripe rust and powdery mildew disease. The effectiveness of seedling resistance against each pathogen correlated with the level of transgene expression. No evidence of accelerated leaf necrosis or up‐regulation of senescence gene markers was apparent in these seedlings, suggesting senescence is not required for Lr34 resistance, although leaf tip necrosis occurred in mature plant flag leaves. Several abiotic stress‐response genes were up‐regulated in these seedlings in the absence of rust infection as previously observed in adult plant flag leaves of hexaploid wheat. Increasing day length significantly increased Lr34 seedling resistance. These data demonstrate that expression of a highly durable, broad‐spectrum adult plant resistance gene can be modified to provide seedling resistance in durum wheat.     

Genetic analysis and location of a resistance gene for <Emphasis Type="BoldItalic">Puccinia striiformis</Emphasis> f. sp. <Emphasis Type="BoldItalic">tritici</Emphasis> in wheat cultivar Zhengmai 7698

Wheat stripe (yellow) rust, caused by Puccinia striiformis West. f. sp. tritici (Pst), is one of the most destructive diseases in many wheat-growing countries, especially in China, the largest stripe rust epidemic area in the world. Growing the resistant cultivars is an effective, economic and environmentally friendly way to control this disease. Wheat cultivar Zhengmai 7698 has shown a high-level resistance to wheat stripe rust. To elucidate its genetic characteristics and location of the resistance gene, Zhengmai 7698 was crossed with susceptible variety Taichung 29 to produce \(\hbox {F}_{{1}}\), \(\hbox {F}_{{2}}\) and \(\hbox {BC}_{{1}}\) progeny generations. The genetic analysis showed that the stripe rust resistance in Zhengmai 7698 to Pst predominant race CYR32 was controlled by a single-dominant gene, namedYrZM. Bulked segregant analysis and simple sequence repeat (SSR) markers were used to map the gene. Four SSR markers, Xbarc198, Xwmc179, Xwmc786 and Xwmc398 on chromosome 6BL were polymorphic between the parents and resistance, and susceptible bulks. A linkage genetic map was constructed using 212 \(\hbox {F}_{{2}}\) plants in the sequential order of Xwmc398, Xwmc179, YrZM, Xbarc198, Xwmc786. As this gene is effective against predominant race CYR32, it is useful in combination with other resistance genes for developing new wheat cultivars with resistance to stripe rust.     

Genetic analysis and location of gene for resistance to stripe rust in wheat international differential host Strubes Dickkopf

Strubes Dickkopf is the sixth differential in the world set for wheat stripe (yellow) rust. It is very important to clarify its genetic character of resistance to stripe rust and to develop the molecular markers linked to resistance genes. The NIL Taichung 29*6/Strubes Dickkopf, which was obtained by Strubes Dickkopf as the gene donor and Taichung 29 as the genetic background through backcross breeding, was crossed with the recurrent parent Taichung 29, inbred, and backcrossed to obtain the F₁, F₂ and BC₁ population. The genetic analysis of the cross Taichung 29/(Taichung 29*6/Strubes Dickkopf) was assessed by inoculating the rust race CYR26 at seedling stage. Bulked segregant analysis (BSA) and F₂ segregation analysis were used for detecting polymorphic primers to locate the gene. The resistance of the NIL Taichung 29*6/Strubes Dickkopf to CYR26 was controlled by a single dominant gene, named YrSD. The primer pair Xbarc59 on 5B was linked to YrSD and the genetic distance between Xbarc59 and YrSD was 2.4 cM. The molecular marker Xbarc59 closely linked to the gene YrSD could be used in marker-assisted selection for resistance to stripe rust in wheat breeding programmes.     

Molecular characterization of a wheat–Psathyrostachys huashanica Keng 2Ns disomic addition line with resistance to stripe rust

We characterized a wheat–Psathyrostachys huashanica derived line 3-6-4-1 based on genomic in situ hybridization (GISH), molecular marker analysis, and agronomic trait evaluations. The GISH investigations showed that the 3-6-4-1 contained 42 wheat chromosomes and a pair of P. huashanica chromosomes. The homoeologous relationships of the introduced P. huashanica chromosomes were determined using EST-STS multiple loci markers from seven wheat homoeologous groups in the parents and the addition line. Twelve EST-STS markers located on the homoeologous group 2 chromosomes of wheat amplified polymorphic bands in 3-6-4-1, which were unique to P. huashanica. An introduced Ns chromosome pair that belonged to homoeologous group 2 was identified using chromosome-specific markers. Inoculation with isolates of the stripe rust pathotypes, CYR31, CYR32, and SY11-14, and mixed races (CYR31, CYR32, and SY11-14) in the seeding and adult stage, respectively, showed that 3-6-4-1 was generally resistant to stripe rust, which was probably attributable to its P. huashanica parent. We also compared a complete set of wheat–P. huashanica disomic addition lines (1Ns–7Ns, 2n = 44 = 22II) to assess their agronomic traits and morphological characteristics, which showed that 3-6-4-1 had improved spike traits compared with its parents. The P. huashanica 2Ns chromosome-specific molecular markers in 3-6-4-1 could be useful for marker-assisted selection in breeding programs to combat stripe rust. This line can be used as a donor source to introduce novel excellent genes from P. huashanica into wheat to widen its genetic diversity, thereby providing new germplasms for wheat breeding.     

Mapping of stripe rust resistance gene in an <Emphasis Type="Italic">Aegilops caudata</Emphasis> introgression line in wheat and its genetic association with leaf rust resistance

A pair of stripe rust and leaf rust resistance genes was introgressed from Aegilops caudata, a nonprogenitor diploid species with the CC genome, to cultivated wheat. Inheritance and genetic mapping of stripe rust resistance gene in backcross-recombinant inbred line (BC-RIL) population derived from the cross of a wheat–Ae. caudata introgression line (IL) T291-2(pau16060) with wheat cv. PBW343 is reported here. Segregation of BC-RILs for stripe rust resistance depicted a single major gene conditioning adult plant resistance (APR) with stripe rust reaction varying from TR-20MS in resistant RILs signifying the presence of some minor genes as well. Genetic association with leaf rust resistance revealed that two genes are located at a recombination distance of 13%. IL T291-2 had earlier been reported to carry introgressions on wheat chromosomes 2D, 3D, 4D, 5D, 6D and 7D. Genetic mapping indicated the introgression of stripe rust resistance gene on wheat chromosome 5DS in the region carrying leaf rust resistance gene LrAc, but as an independent introgression. Simple sequence repeat (SSR) and sequence-tagged site (STS) markers designed from the survey sequence data of 5DS enriched the target region harbouring stripe and leaf rust resistance genes. Stripe rust resistance locus, temporarily designated as YrAc, mapped at the distal most end of 5DS linked with a group of four colocated SSRs and two resistance gene analogue (RGA)-STS markers at a distance of 5.3 cM. LrAc mapped at a distance of 9.0 cM from the YrAc and at 2.8 cM from RGA-STS marker Ta5DS_2737450, YrAc and LrAc appear to be the candidate genes for marker-assisted enrichment of the wheat gene pool for rust resistance.     

两系杂交小麦恢复系MR168抗条锈病基因遗传分析及分子标记定位

小麦条锈病是影响杂交小麦普及推广的重要因素。文章利用基因推导法和SSR分子标记技术,研究了温光型两系杂交小麦恢复系MR168的抗条锈性遗传规律及其控制基因染色体位置。结果表明,MR168对CY29、CY31、CY32、CY33等条锈菌生理小种表现高抗至免疫;对SY95-71/MR168杂交组合的正反交F1、BC1、F2和F3群体分单株接种鉴定显示,MR168对CY32号小种的抗性受1对显性核基因控制,该抗病基因来源于春小麦品种辽春10号。利用集群分离分析法(Bulked segregant analysis,BSA)和简单重复序列(Simple sequence repeat,SSR)分子标记分析抗病亲本MR168、感病亲本SY95-71及183个F2代单株,发现了与MR168抗条锈病基因连锁的5个微卫星标记Xgwm273、Xgwm18、Xbarc187、Xwmc269、Xwmc406,并将该基因初步定位在1BS着丝粒附近,暂命名为YrMR168;构建了包含YrMR168的SSR标记遗传图谱,距离YrMR168最近的两个微卫星位点是Xgwm18和Xbarc187,遗传距离分别为1.9 cM和2.4 cM,这两个微卫星标记可用于杂交小麦抗条锈病分子标记辅助育种。     

Remapping of the stripe rust resistance gene <Emphasis Type="Italic">Yr10</Emphasis> in common wheat

Key message

Yr10 is an important gene to control wheat stripe rust, and the search for Yr10 needs to be continued.

Abstract

Wheat stripe rust or yellow rust is a devastating fungal disease caused by Puccinia striiformis f. sp. tritici (Pst). Host disease resistance offers a primary source for controlling wheat stripe rust. The stripe rust resistance gene Yr10 confers the race-specific resistance to most tested Pst races in China including CYR29. Early studies proposed that Yr10 was a nucleotide-binding site, leucine-rich repeat gene archived as GenBank accession AF149112 (hereafter designated the Yr10 candidate gene or Yr10 _CG ). In this study, we revealed that 15 Chinese wheat cultivars positive for Yr10 _CG are susceptible to CYR29. We then expressed the Yr10 _CG cDNA in the common wheat ‘Bobwhite’. The Yr10 _CG -cDNA positive transgenic plants were also susceptible to CYR29. Thus, it is highly unlikely that Yr10 _CG corresponds to the Yr10 resistance gene. Using the Yr10 donor ‘Moro’ and the Pst-susceptible wheat ‘Huixianhong’, we generated two F₃ populations that displayed a single Mendelian segregation on the Yr10 gene, and used them to remap the Yr10 gene. Six markers were placed in the Yr10 region, with the Yr10 _CG gene now mapping about 1.2-cM proximal to the Yr10 locus and the Xsdauw79 marker is completely linked to the Yr10 locus. Apparently, the Yr10 gene has not yet been identified. Fine mapping and positional cloning of Yr10 is important for gene pyramiding for stripe rust resistance in wheat.

     

Molecular mapping of a non-host resistance gene YrpstY1 in barley (Hordeum vulgare L.) for resistance to wheat stripe rust

Cultivated barley (Hordeum vulgare L.) is considered as a non-host or inappropriate host species for wheat stripe rust caused by Puccinia striiformis f. sp. tritici. Most barley cultivars show a broad-spectrum resistance to wheat stripe rust. To determine the genes for resistance to wheat stripe rust in barley, a cross was made between a resistant barley line Y12 and a susceptible line Y16. The two parents, F(1) and 147 BC(1) plants were tested at seedling stage with Chinese prevalent race CYR32 of Puccinia striiformis f. sp. tritici by artificial inoculation in greenhouse. The results indicated that Y12 possessed one dominant resistance gene to wheat stripe rust, designated YrpstY1 provisionally. A total of 388 simple sequence repeat (SSR) markers were used to map the resistance gene in Y12 using bulked segregant analysis. A linkage map, including nine SSR loci on chromosome 7H and YrpstY1, was constructed using the BC(1) population, indicating that the resistance gene YrpstY1 is located on chromosome 7H. It is potential to transfer the resistance gene into common wheat for stripe rust resistance.     

Molecular mapping of stripe rust resistance gene <Emphasis Type="Italic">YrHu</Emphasis> derived from <Emphasis Type="Italic">Psathyrostachys huashanica</Emphasis>

Wheat stripe rust is a destructive disease that affects most wheat-growing areas worldwide. Resistance genes from related species and genera add to the genetic diversity available to wheat breeding programs. The stripe rust-resistant introgression line H9020-17-25-6-4 was developed from a cross of resistant Psathyrostachys huashanica with the susceptible wheat cultivar 7182. H9020-17-25-6-4 is resistant to all existing Chinese stripe rust races, including the three most widely virulent races, CYR32, CYR33, and V26. We attempted to characterize this new line by genomic in situ hybridization (GISH) and genetic analysis. GISH using P. huashanica genomic DNA as a probe indicated that the translocated segment was too small to be detected. Genetic analysis involving F₁, F₂, and F_2:3 materials derived from a cross of Mingxian 169 and H9020-17-25-6-4 indicated that a single dominant gene from H9020-17-25-6-4, temporarily designated YrHu, conferred resistance to CYR29 and CYR33. A genetic map consisting of four simple sequence repeat, two sequence-tagged site (STS), and two sequence-related amplified polymorphism markers was constructed. YrHu was located on the short arm of chromosome 3A and was about 0.7 and 1.5 cM proximal to EST-STS markers BG604577 and BE489244, respectively. Both the gene and the closely linked markers could be used in marker-assisted selection.     

Histological and cytological characterization of adult plant resistance to wheat stripe rust

Wheat cultivar Xingzi 9104 (XZ) possesses adult plant resistance (APR) to stripe rust caused by Puccinia striiformis f. sp. tritici (Pst). In this study, histological and cytological experiments were conducted to elucidate the mechanisms of APR in XZ. The results of leaf inoculation experiments indicated that APR was initiated at the tillering stage, gradually increased as the plant aged and highly expressed after boot stage. The histology and oxidative burst in infected leaves of plants at seedling, tillering and boot stages were examined using light microscopic and histochemical methods. Subcellular changes in the host–pathogen interactions during the seedling and boot stages were analyzed by transmission electron microscopy. The results showed that haustorium formation was retarded in the adult plants and that the differentiation of secondary intercellular hyphae was significantly inhibited, which decreased the development of microcolonies in the adult plants, especially in plants of boot stage. The expression of APR to stipe rust during wheat development was clearly associated with extensive hypersensitive cell death of host cells and localized production of reactive oxygen species, which coincided with the restriction of fungal growth in infection sites in adult plants. At the same time, cell wall-related resistance in adult plants prevented ingression of haustorial mother cells into plant cells. Haustorium encasement was coincident with malformation or death of haustoria. The results provide useful information for further determination of mechanisms of wheat APR to stripe rust. Key message The expression of APR to stipe rust in wheat cultivar Xingzi 9104 (XZ) was clearly associated with extensive hypersensitive cell death of host cells and the localized production of reactive oxygen species.     

Identification and genetic mapping of a recessive gene for resistance to stripe rust in wheat line LM168-1

Stripe rust (or yellow rust), caused by the fungus Puccinia striiformis f. sp. tritici (Pst), is one of the most important foliar diseases of wheat. Characterization and utilization of novel resistant genes is the most effective, economic and environmentally friendly approach to controlling the disease. Wheat line LM168-1, which was derived from a cross between common wheat Chuannong 16 and Milan, has good adult-plant resistance to stripe rust, based on field tests over several years. To elucidate the genetic basis of resistance, LM168-1 was crossed with susceptible variety SY95-71. Parents and F1, F2, BC1 and F2:3 progenies were tested in 2009–2011 in a field inoculated with the predominant races of Pst in China. The genetic analysis showed that resistance to stripe rust in LM168-1 was controlled by a single recessive gene, temporarily designated yrLM168. Simple sequence repeat (SSR), resistance gene analog polymorphism (RGAP) and target region amplification polymorphism (TRAP) techniques were used to identify molecular markers linked to the resistance locus. Finally, a linkage group consisting of two SSR, four RGAP and five TRAP markers was constructed for yrLM168 with 102 F2 plants. The closest markers R1 and R2 flanked the resistance gene locus at 2.4 and 2.4 cM, respectively. Furthermore, two SSR markers Xwmc59 and Xwmc145 assigned the gene to chromosome 6A. Because yrLM168 confers high-level resistance to the predominant races of Pst in China, it should be useful in stripe rust resistance breeding programs. The closely linked markers can be used for rapidly transferring yrLM168 to wheat breeding populations.     

Genetic mapping of a putative Thinopyrum intermedium-derived stripe rust resistance gene on wheat chromosome 1B

Key message

Stripe rust resistance transferred from Thinopyrum intermedium into common wheat was controlled by a single dominant gene, which mapped to chromosome 1B near Yr26 and was designated YrL693.

Abstract

Stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is a highly destructive disease of wheat (Triticum aestivum). Stripe rust resistance was transferred from Thinopyrum intermedium to common wheat, and the resulting introgression line (L693) exhibited all-stage resistance to the widely virulent and predominant Chinese pathotypes CYR32 and CYR33 and to the new virulent pathotype V26. There was no cytological evidence that L693 had alien chromosomal segments from Th. intermedium. Genetic analysis of stripe rust resistance was performed by crossing L693 with the susceptible line L661. F₁, F₂, and F_2:3 populations from reciprocal crosses showed that resistance was controlled by a single dominant gene. A total 479 F_2:3 lines and 781 pairs of genomic simple sequence repeat (SSR) primers were employed to determine the chromosomal location of the resistance gene. The gene was linked to six publicly available and three recently developed wheat genomic SSR markers. The linked markers were localized to wheat chromosome 1B using Chinese Spring nulli-tetrasomic lines, and the resistance gene was localized to chromosome 1B based on SSR and wheat genomic information. A high-density genetic map was also produced. The pedigree, molecular marker data, and resistance response indicated that the stripe rust resistance gene in L693 is a novel gene, which was temporarily designated YrL693. The SSR markers that co-segregate with this gene (Xbarc187-1B, Xbarc187-1B-1, Xgwm18-1B, and Xgwm11-1B) have potential application in marker-assisted breeding of wheat, and YrL693 will be useful for broadening the genetic basis of stripe rust resistance in wheat.     

黄淮麦区小麦品种(系)中Yr26基因的SSR检测

选用与Yr26紧密连锁的SSR标记Xgwm11和Xgwm18结合田间抗性鉴定,对239份黄淮麦区小麦品种(系)进行检测,以明确Yr26基因在黄淮麦区小麦品种资源中的分布.结果表明:共有35份品种(系)含有与Yr26紧密连锁的SSR标记Xgwm18或Xgwm11的特征带,占检测样本的14.6%.在这35份材料中,31份田间抗性鉴定表现免疫至中抗,4份表现中感.分子标记检测与田间抗病性检测吻合度较好,该标记可以用于Yr26基因的分子标记辅助选择.综合分子标记和田间鉴定,31份小麦(系)含有Yr26基因,占102份抗病材料的30.39%.