Aizhong Cao

Serine/threonine kinase gene Stpk-V, a key member of powdery mildew resistance gene Pm21, confers powdery mildew resistance in wheat

Powdery mildew resistance gene Pm21, located on the chromosome 6V short arm of Haynaldia villosa and transferred to wheat as a 6VS·6AL translocation (T6VS·6AL), confers durable and broad-spectrum resistance to wheat powdery mildew. Pm21 has become a key gene resource for powdery mildew resistance breeding all over the world. In China, 12 wheat varieties containing Pm21 have been planted on more than 3.4 million hectares since 2002. Pm21 has been intractable to molecular genetic mapping because the 6VS does not pair and recombine with the 6AS. Moreover, all known accessions of H. villosa are immune to powdery mildew fungus. Pm21 is still defined by cytogenetics as a locus. In the present study, a putative serine and threonine protein kinase gene Stpk-V was cloned and characterized with an integrative strategy of molecular and cytogenetic techniques. Stpk-V is located on the Pm21 locus. The results of a single cell transient expression assay showed that Stpk-V could decrease the haustorium index dramatically. After the Stpk-V was transformed into a susceptible wheat variety Yangmai158, the characterized transgenic plants showed high and broad-spectrum powdery mildew resistance similar to T6VS·6AL. Silencing of the Stpk-V by virus-induced gene silencing in both T6VS·6AL and H. villosa resulted in their increased susceptibility. Stpk-V could be induced by Bgt and exogenous H2O2, but it also mediated the increase of endogenous H2O2, leading to cell death and plant resistance when the plant was attacked by Bgt.

Pm55, a developmental-stage and tissue-specific powdery mildew resistance gene introgressed from Dasypyrum villosum into common wheat

Key messagePowdery mildew resistance gene Pm55 was physically mapped to chromosome arm 5VS FL 0.60–0.80 of Dasypyrum villosum. Pm55 is present in T5VS·5AL and T5VS·5DL translocations, which should be valuable resources for wheat improvement.AbstractPowdery mildew caused by Blumeria graminis f. sp. tritici is a major wheat disease worldwide. Exploiting novel genes effective against powdery mildew from wild relatives of wheat is a promising strategy for controlling this disease. To identify novel resistance genes for powdery mildew from Dasypyrum villosum, a wild wheat relative, we evaluated a set of Chinese Spring-D. villosum disomic addition and whole-arm translocation lines for reactions to powdery mildew. Based on the evaluation data, we concluded that the D. villosum chromosome 5V controls post-seedling resistance to powdery mildew. Subsequently, three introgression lines were developed and confirmed by molecular and cytogenetic analysis following ionizing radiation of the pollen of a Chinese Spring-D. villosum 5V disomic addition line. A homozygous T5VS·5AL translocation line (NAU421) with good plant vigor and full fertility was further characterized using sequential genomic in situ hybridization, C-banding, and EST-STS marker analysis. A dominant gene permanently named Pm55 was located in chromosome bin 5VS 0.60–0.80 based on the responses to powdery mildew of all wheat-D. villosum 5V introgression lines evaluated at both seeding and adult stages. This study demonstrated that Pm55 conferred growth-stage and tissue-specific dependent resistance; therefore, it provides a novel resistance type for powdery mildew. The T5VS·5AL translocation line with additional softness loci Dina/Dinb of D. villosum provides a possibility of extending the range of grain textures to a super-soft category. Accordingly, this stock is a new source of resistance to powdery mildew and may be useful in both resistance mechanism studies and soft wheat improvement.

Transformation of Wheat Thaumatin-Like Protein Gene and Analysis of Reactions to Powdery Mildew and Fusarium Head Blight in Transgenic Plants

Abstract A thaumatin-like protein gene, Ta-Tlp , was cloned from wheat in previous studies, which was expressed in a high level in wheat ( Triticum aestivum L.) 6VS/6AL translocation line with high resistance to powdery mildew ( Erysiphe graminis f.sp. tritici Em. Marchal.), implying its close relation to the resistance of the disease. To further understand the genes function, Ta-Tlp was constructed into an expression vector driven by the strong ubi promoter. The vector pAHC-TlP constructed was transformed into immature embryo-derived calli of common wheat cultivar Yangmai 158 through particle bombardment. After two rounds of herbicide bialaphos selection and regeneration, herbicide-resistance plants were obtained. The Ta-Tlp proved to be integrated into the wheat genome and was expressed in T 1 and T 2 generations by PCR, Southern blot, and RT-PCR analysis. The transgenic plants of T 0 , T 1 , and T 2 generations were inoculated by E. graminis and Fusarium graminearum for resistance identification. All plants of T 0 , T 1 , and T 2 generations were resistant to wheat powdery mildew by delaying disease development, but no distinct resistance to Fusarium head blight.

RLP1.1, a novel wheat receptor-like protein gene, is involved in the defence response against Puccinia striiformis f. sp. tritici

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most serious diseases of wheat; therefore, exploring effective resistance-related genes is critical for breeding and studying resistance mechanisms. However, only a few stripe rust resistance genes and defence-related genes have been cloned. Moreover, transgenic wheat with enhanced stripe rust resistance has rarely been reported. Receptor-like proteins (RLPs) are known to be involved in defence and developmental pathways. In this research, a novel RLP gene TaRLP1.1 was characterized as an important stripe rust defence gene. TaRLP1.1 was screened by GeneChip and was found to be induced by Pst specifically in the resistant variety. Knock down of TaRLP1.1 in the stripe rust-resistant plants resulted in increased susceptibility to Pst, and phenolic autofluorogen accumulation at the pathogen–host interaction sites, usually correlated with the hypersensitive response, was decreased dramatically. However, when the TaRLP1.1 gene was transformed into the susceptible wheat variety Yangmai158, the transgenic plants showed highly increased resistance to Pst, and the hypersensitive response was enhanced at the infection sites. Meanwhile, the expression of pathogenesis-related genes decreased in the TaRLP1.1-silenced plants and increased in the TaRLP1.1-overexpressing plants. Thus, it was proposed that TaRLP1.1 greatly contributed to the hypersensitive response during the pathogen–host interaction. Along with the functional analysis, an evolutionary study of the TaRLP1 family was performed. Characterization of TaRLP1.1 may facilitate breeding for stripe rust resistance and better understanding of the evolution of the RLP genes in wheat.

Screening and application of EST-based PCR markers specific to individual chromosomes of Haynaldia villosa.

Abstract In our previous studies, a series of wheat– Haynaldia villosa alien chromosome lines including translocation lines were developed through pollen-irradiation induction. To identify H. villosa chromosomes or chromosome segments in these lines, 240 sequence tagged site (STS) primer pairs were designed based on expressed sequence tags (ESTs) of rice ( Oryza sativa L.) and wheat, and 34 of them amplified specific polymorphic bands between T. aestivum cv. Chinese Spring and H. villosa . These 34 STS primer pairs were further used for screening markers specific to individual chromosomes of H. villosa using a complete set of disomic addition lines. Marker CINAU32 −300 could be used for tracing chromosome 1V, markers CINAU33 −280 , CINAU34 −510 , CINAU35 −1100 , CINAU36 −380 , and CINAU37 −400 for 2V, marker CINAU38 −250 for 3V, markers CINAU39 −950 , and CINAU40 −800 for 4V, markers CINAU41 −745 and CINAU42 −1050 for 5V, and markers CINAU44 −765 and CINAU45 −495 for 7V. Chromosomes and chromosome segments from H. villosa in the backcrossed generations derived from pollen irradiation were characterized using these EST-STS markers combined with two 6V specific markers that developed in earlier studies. A complete set of 1V to 7V addition lines and 18 translocation lines involving different H. villosa chromosomes were identified. Therefore, these chromosome-specific EST-STS markers are useful for detecting H. villosa chromosomes and chromosome segments in common wheat background.

Characterization of T.aestivum-H.californicum chromosome addition lines DA2H and MA5H

In order to transfer useful genes of Hordeum californicum into common wheat (Triticum aestivum L.), the T. aestivum c.v. Chinese Spring (CS)-H. californicum amphiploid was crossed to CS, and its backcrossing and self-fertilized progenies were analyzed by morphological observation, cytological, biochemical and molecular marker techniques. Alien addition lines with two H. californicum chromosomes were identified and their genetic constitution was characterized. STS-PCR analysis using chromosome 2B specific markers indicated that chromosome H3 of H. californicum belongs to homologous group 2, and was thus designated 2H. SDS-PAGE showed that chromosome H2 of H. californicum belongs to homologous group 5, and was designated 5H. The CS-H. californicum amphiploid and the chromosome addition lines (DA2H and MA5H) identified were evaluated for powdery mildew (Erysiphe graminis f. sp. triticii) resistance in field. The preliminary results indicated that the amphiploid showed higher powdery mildew resistance than CS. However, chromosome addition lines DA2H and MA5H were highly susceptible to powdery mildew, indicating that major powdery mildew resistant genes of H. californicum should be located on chromosomes other than 2H and 5H.

The Hv-SGT1 Gene from Haynaldia villosa Contributes to Resistances Towards Both Biotrophic and Hemi-Biotrophic Pathogens in Common Wheat (Triticum aestivum L.)

The SGT1 protein is essential for R protein-mediated and PAMPs-triggered resistance in many plant species. Here we reported the isolation and characterization of the Hv-SGT1 gene from Haynaldia villosa (2n = 14, VV). Analysis of the subcellular location of Hv-SGT1 by transient expression of a fusion to GFP indicated its presence in the cytoplasm and nucleus. Levels of Hv-SGT1 transcripts were increased by inoculation with either the biotrophic pathogen Blumeria graminis DC. f. Sp. tritici (Bgt) or the hemi-biotrophic pathogen Fusarium graminearum (Fg). Levels of Hv-SGT1 showed substantial increase following treatment with H2O2 and methyl jasmonate (MeJA), only slightly induced following exposure to ethephon or abscisic acid, but not changed following exposure to salicylic acid. The demonstration that silencing of Hv-SGT1 substantially reduced resistance to Bgt indicated that Hv-SGT1 was an essential component of disease resistance in H . villosa . The over-expression of Hv-SGT1 in Yangmai 158 enhanced resistance to powdery mildew, and this correlated with increased levels of whole-cell reactive oxygen intermediates at the sites of penetration by the pathogens. Compared with wild-type plants, the expression levels of genes related to the H2O2 and JA signaling pathways were lower in the Hv-SGT1 silenced plants and higher in the Hv-SGT1 over-expressing plants. Therefore, the involvement of Hv-SGT1 in H2O2 production correlates with the hypersensitive response and jasmonic acid signaling. Our novel demonstration that wheat with over-expressed Hv-SGT1 showed enhanced resistance to both powdery mildew and FHB suggests that it could served as a transgenic genetic resource in wheat breeding for multiple disease resistance.

The role of wheat jasmonic acid and ethylene pathways in response to Fusarium graminearum infection

To study the roles of jasmonic acid (JA) and ethylene (ET) pathways in mediating defense against wheat Fusarium head blight (FHB), the expression patterns of genes in the Fusarium graminearum-challenged spikes between Wangshuibai and its susceptible mutant NAUH117 at the Fhb1 locus were compared using wheat microarray. The results showed that most of JA-associated genes were induced in Wangshuibai while only a few were induced in NAUH117, and most ET-associated genes were up-regulated in both genotypes. ELISA assay showed that in the F. graminearum-challenged spikes, endogenous JA content was increased in Wangshuibai while not in NAUH117. Pre-treatment with exogenous methyl JA could decrease the wheat disease severity. However, pretreatment by exogenous ethephon had no such effect. A lipid transfer protein gene, which is a representative gene for JA pathway, was selected for function analysis in Arabidopsis system using a T-DNA insertion mutant line for LTP gene. It was found that the mutant showed compromised FHB resistance compared with its wildtype, proving the possible role of LTP in FHB resistance of Arabidopsis. These results demonstrated that JA pathway should play a critical role in FHB resistance.

A fast-neutron induced chromosome fragment deletion of 3BS in wheat landrace Wangshuibai increased its susceptibility to Fusarium head blight

Fusarium head blight (FHB), also called wheat scab, is an important disease in warm and humid regions worldwide, which not only reduces crop yield and grain quality, but also is a major safety concern in food and feed production due to mycotoxin contamination. Growing wheat cultivars with FHB resistance is one of the most economical and effective means to control the disease. Chinese wheat landrace Wangshuibai is an important resistant source from southern China. Several resistance QTLs in Wangshuibai were identified and mapped on chromosomes or chromosomal arms including 3BS, 4B, 6BS, 7AL, etc. In the present research, a mutant with increased FHB susceptibility, designated as NAUH117, was identified from the M1 progenies of Wangshuibai irradiated by fast neutron. Genetic analysis of the F1, F2, and F2:3 families from the reciprocal cross of Wangshuibai and NAUH117 indicated that NAUH117 was a recessive mutant. Genome-wide molecular marker analysis identified a deletion in the short arm of chromosome 3B of NAUH117, spanning the region of FL0.57 to FL1.00 that covers the locus of Fhb1 previously mapped on chromosome 3BS. Further molecular cytogenetics characterization by bi-color fluorescence in situ hybridization using three repetitive sequences, pSc119.2, pAs1 and GAA-satellite indicated that a multiple chromosome rearrangements occurred in chromosomes 3B, 6B, 3D, 4D, and 3A of the mutant. During these processes, a distal fragment of chromosome arm 3BS was eliminated, which is confirmed by molecular marker analysis. Four markers covered the deletion fragment were used for analysis of the F2 population. The result showed that the 3BS deletion was only present in the susceptible plants, indicating that the deletion of 3BS fragment in NAUH117 increased susceptibility to FHB. The susceptible mutant will be valuable for the validation of the contribution of the resistant QTL located on 3BS, and for the characterization of the molecular mechanisms of FHB resistance in Wangshuibai.

Two members of TaRLK family confer powdery mildew resistance in common wheat

BackgroundPowdery mildew, caused by Blumeria graminearum f.sp. tritici (Bgt), is one of the most severe fungal diseases of wheat. The exploration and utilization of new gene resources is the most effective approach for the powdery mildew control.ResultsWe report the cloning and functional analysis of two wheat LRR-RLKs from T. aestivum c.v. Prins- T. timopheevii introgression line IGV1-465, named TaRLK1 and TaRLK2, which play positive roles in regulating powdery mildew resistance in wheat. The two LRR-RLKs contain an ORF of 3,045 nucleotides, encoding a peptide of 1014 amino acids, with seven amino acids difference. Their predicted proteins possess a signal peptide, several LRRs, a trans-membrane domain, and a Ser/Thr protein kinase domain. In response to Bgt infection, the TaRLK1/2 expression is up-regulated in a developmental-stage-dependent manner. Single-cell transient over-expression and gene-silencing assays indicate that both genes positively regulate the resistance to mixed Bgt inoculums. Transgenic lines over-expressing TaRLK1 or TaRLK2 in a moderate powdery mildew susceptible wheat variety Yangmai 158 led to significantly enhanced powdery mildew resistance. Exogenous applied salicylic acid (SA) or hydrogen peroxide (H2O2) induced the expression of both genes, and H2O2 had a higher accumulation at the Bgt penetration sites in RLK over-expression transgenic plants, suggesting a possible involvement of SA and altered ROS homeostasis in the defense response to Bgt infection. The two LRR-RLKs are located in the long arm of wheat chromosome 2B, in which the powdery mildew resistance gene Pm6 is located, but in different regions.ConclusionsTwo members of TaRLK family were cloned from IGV1-465. TaRLK1 and TaRLK2 contribute to powdery mildew resistance of wheat, providing new resistance gene resources for wheat breeding.