Overexpression of defense response genes in transgenic wheat enhances resistance to Fusarium head blight

Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum and other Fusarium species, is a major disease problem for wheat production worldwide. To combat this problem, large-scale breeding efforts have been established. Although progress has been made through standard breeding approaches, the level of resistance attained is insufficient to withstand epidemic conditions. Genetic engineering provides an alternative approach to enhance the level of resistance. Many defense response genes are induced in wheat during F. graminearum infection and may play a role in reducing FHB. The objectives of this study were (1) to develop transgenic wheat overexpressing the defense response genes α-1-purothionin, thaumatin-like protein 1 (tlp-1), and β-1,3-glucanase; and (2) to test the resultant transgenic wheat lines against F. graminearum infection under greenhouse and field conditions. Using the wheat cultivar Bobwhite, we developed one, two, and four lines carrying the α-1-purothionin, tlp-1, and β-1,3-glucanase transgenes, respectively, that had statistically significant reductions in FHB severity in greenhouse evaluations. We tested these seven transgenic lines under field conditions for percent FHB disease severity, deoxynivalenol (DON) mycotoxin accumulation, and percent visually scabby kernels (VSK). Six of the seven lines differed from the nontransgenic parental Bobwhite line for at least one of the disease traits. A β-1,3-glucanase transgenic line had enhanced resistance, showing lower FHB severity, DON concentration, and percent VSK compared to Bobwhite. Taken together, the results showed that overexpression of defense response genes in wheat could enhance the FHB resistance in both greenhouse and field conditions.     

抗茎腐病转基因小麦新种质的筛选

为拓宽小麦茎腐病(又称茎基腐病)抗源种类,筛选抗茎腐病小麦新种质,对43份转TaPIMP1、AtNPR1和Gastrodianin基因小麦纯合株系,进行目的基因表达分析,以及茎腐病、纹枯病和赤霉病抗性鉴定。结果表明,转基因株系的目的基因均能正常表达;转基因株系间茎腐病抗性差异明显,24份转基因株系茎腐病抗性,比受体对照扬麦12显著提高;转基因株系茎腐病抗性与纹枯病抗性相关性显著,与赤霉病相关性不显著。结合农艺性状鉴定,筛选出5份抗茎腐病转基因株系,其中2份兼抗纹枯病和赤霉病,1份兼抗纹枯病,可作为长江中下游麦区茎腐病备用抗源。     

Tissue‐specific and pathogen‐inducible expression of a fusion protein containing a Fusarium‐specific antibody and a fungal chitinase protects wheat against Fusarium pathogens and mycotoxins

Fusarium head blight (FHB) in wheat and other small grain cereals is a globally devastating disease caused by toxigenic Fusarium pathogens. Controlling FHB is a challenge because germplasm that is naturally resistant against these pathogens is inadequate. Current control measures rely on fungicides. Here, an antibody fusion comprised of the Fusarium spp.‐specific recombinant antibody gene CWP2 derived from chicken, and the endochitinase gene Ech42 from the biocontrol fungus Trichoderma atroviride was introduced into the elite wheat cultivar Zhengmai9023 by particle bombardment. Expression of this fusion gene was regulated by the lemma/palea‐specific promoter Lem2 derived from barley; its expression was confirmed as lemma/palea‐specific in transgenic wheat. Single‐floret inoculation of independent transgenic wheat lines of the T₃ to T₆ generations revealed significant resistance (type II) to fungal spreading, and natural infection assays in the field showed significant resistance (type I) to initial infection. Gas chromatography–mass spectrometry analysis revealed marked reduction of mycotoxins in the grains of the transgenic wheat lines. Progenies of crosses between the transgenic lines and the FHB‐susceptible cultivar Huamai13 also showed significantly enhanced FHB resistance. Quantitative real‐time PCR analysis revealed that the tissue‐specific expression of the antibody fusion was induced by salicylic acid drenching and induced to a greater extent by F. graminearum infection. Histochemical analysis showed substantial restriction of mycelial growth in the lemma tissues of the transgenic plants. Thus, the combined tissue‐specific and pathogen‐inducible expression of this Fusarium‐specific antibody fusion can effectively protect wheat against Fusarium pathogens and reduce mycotoxin content in grain.     

Host‐induced gene silencing of an essential chitin synthase gene confers durable resistance to Fusarium head blight and seedling blight in wheat

Fusarium head blight (FHB) and Fusarium seedling blight (FSB) of wheat, caused by Fusarium pathogens, are devastating diseases worldwide. We report the expression of RNA interference (RNAi) sequences derived from an essential Fusarium graminearum (Fg) virulence gene, chitin synthase (Chs) 3b, as a method to enhance resistance of wheat plants to fungal pathogens. Deletion of Chs3b was lethal to Fg; disruption of the other Chs gene family members generated knockout mutants with diverse impacts on Fg. Comparative expression analyses revealed that among the Chs gene family members, Chs3b had the highest expression levels during Fg colonization of wheat. Three hairpin RNAi constructs corresponding to the different regions of Chs3b were found to silence Chs3b in transgenic Fg strains. Co‐expression of these three RNAi constructs in two independent elite wheat cultivar transgenic lines conferred high levels of stable, consistent resistance (combined type I and II resistance) to both FHB and FSB throughout the T₃ to T₅ generations. Confocal microscopy revealed profoundly restricted mycelia in Fg‐infected transgenic wheat plants. Presence of the three specific short interfering RNAs in transgenic wheat plants was confirmed by Northern blotting, and these RNAs efficiently down‐regulated Chs3b in the colonizing Fusarium pathogens on wheat seedlings and spikes. Our results demonstrate that host‐induced gene silencing of an essential fungal chitin synthase gene is an effective strategy for enhancing resistance in crop plants under field test conditions.     

A model wheat cultivar for transformation to improve resistance to Fusarium Head Blight

Fusarium head blight (FHB), caused primarily by Fusarium graminearum, is a major disease problem in wheat (Triticum aestivum). Genetic engineering holds significant potential to enhance FHB resistance in wheat. Due to the requirement of screening for FHB resistance on flowers at anthesis, the number of screens carried out in a year is limited. Our objective was to evaluate the feasibility of using the rapid-maturing dwarf wheat cultivar Apogee as an alternative genotype for transgenic FHB resistance research. Our transformation efficiency (number of transgenic plants/number of embryos) for Apogee was 1.33%. Apogee was also found to exhibit high FHB susceptibility and reached anthesis within 4 weeks. Interestingly, microsatellite marker haplotype analysis of the chromosome 3BS FHB resistant quantitative trait locus (QTL) region indicated that this region maybe deleted in Apogee. Our results indicate that Apogee is particularly well suited for accelerating transgenic FHB resistance research and transgenic wheat research in general. C.A. Mackintosh and D.F. Garvin contributed equally to the article and should be considered co-first authors     

Genetically engineered resistance to Fusarium head blight in wheat by expression of Arabidopsis NPR1

Fusarium head blight (FHB) is a devastating disease of wheat and barley which causes extensive losses worldwide. Monogenic, gene-for-gene resistance to FHB has not been reported. The best source of resistance to FHB is a complex, quantitative trait derived from the wheat cv. Sumai 3. Here, we show that the Arabidopsis thaliana NPR1 gene (AtNPR1), which regulates the activation of systemic acquired resistance, when expressed in the FHB-susceptible wheat cv. Bobwhite, confers a heritable, type II resistance to FHB caused by Fusarium graminearum. The heightened FHB resistance in the transgenic AtNPRI -expressing wheat is associated with the faster activation of defense response when challenged by the fungus. PR1 expression is induced rapidly to a high level in the fungus-challenged spikes of the AtNPR1-expressing wheat. Furthermore, benzothiadiazole, a functional analog of salicylic acid, induced PR1 expression faster and to a higher level in the AtNPR1-expressing wheat than in the nontransgenic plants. We suggest that FHB resistance in the AtNPR1-expressing wheat is a result of these plants being more responsive to an endogenous activator of plant defense. Our results demonstrate that NPR1 is an effective candidate for controlling FHB.     

The xylanase inhibitor TAXI‐III counteracts the necrotic activity of a Fusarium graminearum xylanase in vitro and in durum wheat transgenic plants

The xylanase inhibitor TAXI‐III has been proven to delay Fusarium head blight (FHB) symptoms caused by Fusarium graminearum in transgenic durum wheat plants. To elucidate the molecular mechanism underlying the capacity of the TAXI‐III transgenic plants to limit FHB symptoms, we treated wheat tissues with the xylanase FGSG_03624, hitherto shown to induce cell death and hydrogen peroxide accumulation. Experiments performed on lemmas of flowering wheat spikes and wheat cell suspension cultures demonstrated that pre‐incubation of xylanase FGSG_03624 with TAXI‐III significantly decreased cell death. Most interestingly, a reduced cell death relative to control non‐transgenic plants was also obtained by treating, with the same xylanase, lemmas of TAXI‐III transgenic plants. Molecular modelling studies predicted an interaction between the TAXI‐III residue H395 and residues E122 and E214 belonging to the active site of xylanase FGSG_03624. These results provide, for the first time, clear indications in vitro and in planta that a xylanase inhibitor can prevent the necrotic activity of a xylanase, and suggest that the reduced FHB symptoms on transgenic TAXI‐III plants may be a result not only of the direct inhibition of xylanase activity secreted by the pathogen, but also of the capacity of TAXI‐III to avoid host cell death.     

Recognition of glycoside hydrolase 12 proteins by the immune receptor RXEG1 confers Fusarium head blight resistance in wheat

Fusarium head blight (FHB), caused by Fusarium graminearum, is a devastating disease in wheat (Triticum aestivum) that results in substantial yield losses and mycotoxin contamination. Reliable genetic resources for FHB resistance in wheat are lacking. In this study, we characterized glycoside hydrolase 12 (GH12) family proteins secreted by F. graminearum. We established that two GH12 proteins, Fg05851 and Fg11037, have functionally redundant roles in F. graminearum colonization of wheat. Furthermore, we determined that the GH12 proteins Fg05851 and Fg11037 are recognized by the leucine-rich-repeat receptor-like protein RXEG1 in the dicot Nicotiana benthamiana. Heterologous expression of RXEG1 conferred wheat responsiveness to Fg05851 and Fg11037, enhanced wheat resistance to F. graminearum and reduced levels of the mycotoxin deoxynivalenol in wheat grains in an Fg05851/Fg11037-dependent manner. In the RXEG1 transgenic lines, genes related to pattern-triggered plant immunity, salicylic acid, jasmonic acid, and anti-oxidative homeostasis signalling pathways were upregulated during F. graminearum infection. However, the expression of these genes was not significantly changed during infection by the deletion mutant ΔFg05851/Fg11037, suggesting that the recognition of Fg05851/Fg11037 by RXEG1 triggered plant resistance against FHB. Moreover, introducing RXEG1 into three other different wheat cultivars via crossing also conferred resistance to F. graminearum. Expression of RXEG1 did not have obvious deleterious effects on plant growth and development in wheat. Our study reveals that N. benthamiana RXEG1 remains effective when transferred into wheat, a monocot, which in turn suggests that engineering wheat with interfamily plant immune receptor transgenes is a viable strategy for increasing resistance to FHB.     

我国“十三五”育成小麦新品种（系）抗赤霉病进展分析与展望

小麦赤霉病严重威胁我国粮食和食品安全,培育抗赤霉病小麦品种是解决该病害最经济有效的途径。20世纪90年代后,以扬麦158为代表的扬麦、宁麦系列中抗赤霉病品种的育成和大面积推广有效抵御了长江中下游麦区的赤霉病危害,使我国抗赤霉病育种处于国际领先水平。尽管全球明确了7个抗赤霉病基因,为开展抗赤霉病育种提供了重要支撑,但由于赤霉病抗性机制复杂,实现高抗与高产的协调仍极其困难,抗赤霉病仍是当前及未来我国小麦育种的主要目标。对“十三五”期间我国小麦新品系和审定品种的抗性情况以及我国抗赤霉病育种方面取得的进展进行了综述,并提出了重视挖掘和利用扬麦等推广品种中优异抗性基因、将Fhb1导入扬麦等主栽品种的育种技术路线和重视表型精准鉴定等建议,以期为实现我国抗赤霉病育种突破提供借鉴。     

Susceptibility to <Emphasis Type="Italic">Fusarium </Emphasis>head blight is associated with the <Emphasis Type="Italic">Rht-D1b</Emphasis> semi-dwarfing allele in wheat

Fusarium head blight (FHB) is an important disease of wheat worldwide. The cultivar Spark is more resistant than most other UK winter wheat varieties but the genetic basis for this is not known. A mapping population from a cross between Spark and the FHB susceptible variety Rialto was used to identify quantitative trait loci (QTL) associated with resistance. QTL analysis across environments revealed nine QTL for FHB resistance and four QTL for plant height (PH). One FHB QTL was coincident with the Rht-1D locus and accounted for up to 51% of the phenotypic variance. The enhanced FHB susceptibility associated with Rht-D1b is not an effect of PH per se as other QTL for height segregating in this population have no influence on susceptibility. Experiments with near-isogenic lines supported the association between susceptibility and the Rht-D1b allele conferring the semi-dwarf habit. Our results demonstrate that lines carrying the Rht-1Db semi-dwarfing allele are compromised in resistance to initial infection (type I resistance) while being unaffected in resistance to spread within the spike (type II resistance).     

Genetic dissection of a major Fusarium head blight QTL in tetraploid wheat

The devastating effect of Fusarium head blight (FHB) caused by Fusarium graminearum has led to significant financial losses across the Upper Midwest of the USA. These losses have spurred the need for research in biological, chemical, and genetic control methods for this disease. To date, most of the research on FHB resistance has concentrated on hexaploid wheat (Triticum aestivum L.) lines originating from China. Other sources of resistance to FHB would be desirable. One other source of resistance for both hexaploid wheat and tetraploid durum wheat (T. turgidum L. var. durum) is the wild tetraploid, T. turgidum L. var. dicoccoides (T. dicoccoides). Previous analysis of the `Langdon'-T. dicoccoides chromosome substitution lines, LDN(Dic), indicated that the chromosome 3A substitution line expresses moderate levels of resistance to FHB. LDN(Dic-3A) recombinant inbred chromosome lines (RICL) were used to generate a linkage map of chromosome 3A with 19 molecular markers spanning a distance of 155.2 cM. The individual RICL and controls were screened for their FHB phenotype in two greenhouse seasons. Analysis of 83 RICL identified a single major quantitative trait locus, Qfhs.ndsu-3AS, that explains 37% of the phenotypic or 55% of the genetic variation for FHB resistance. A microsatellite locus, Xgwm2, is tightly linked to the highest point of the QTL peak. A region of the LDN (Dic-3A) chromosome associated with the QTL for FHB resistance encompasses a 29.3 cM region from Xmwg14 to Xbcd828.     

Transgenic wheat expressing a barley class II chitinase gene has enhanced resistance against Fusarium graminearum

Fusarium head blight (FHB; scab), primarily caused by Fusarium graminearum, is a devastating disease of wheat worldwide. FHB causes yield reductions and contamination of grains with trichothecene mycotoxins such as deoxynivalenol (DON). The genetic variation in existing wheat germplasm pools for FHB resistance is low and may not provide sufficient resistance to develop cultivars through traditional breeding approaches. Thus, genetic engineering provides an additional approach to enhance FHB resistance. The objectives of this study were to develop transgenic wheat expressing a barley class II chitinase and to test the transgenic lines against F. graminearum infection under greenhouse and field conditions. A barley class II chitinase gene was introduced into the spring wheat cultivar, Bobwhite, by biolistic bombardment. Seven transgenic lines were identified that expressed the chitinase transgene and exhibited enhanced Type II resistance in the greenhouse evaluations. These seven transgenic lines were tested under field conditions for percentage FHB severity, percentage visually scabby kernels (VSK), and DON accumulation. Two lines (C8 and C17) that exhibited high chitinase protein levels also showed reduced FHB severity and VSK compared to Bobwhite. One of the lines (C8) also exhibited reduced DON concentration compared with Bobwhite. These results showed that transgenic wheat expressing a barley class II chitinase exhibited enhanced resistance against F. graminearum in greenhouse and field conditions.     

Development and characterization of wheat- Leymus racemosus translocation lines with resistance to Fusarium Head Blight

Wheat scab (Fusarium Head Blight, FHB) is a destructive disease in the warm and humid wheat-growing areas of the world. Finding diverse sources of FHB resistance is critical for genetic diversity of resistance for wheat breeding programs. Leymus racemosus is a wild perennial relative of wheat and is highly resistant to FHB. Three wheat- L. racemosus disomic addition (DA) lines DA5Lr#1, DA7Lr#1 and DALr.7 resistant to FHB were used to develop wheat- L.racemosus translocation lines through irradiation and gametocidal gene-induced chromosome breakage. A total of nine wheat-alien translocation lines with wheat scab resistance were identified by chromosome C-banding, GISH, telosomic pairing and RFLP analyses. In line NAU614, the long arm of 5Lr#1 was translocated to wheat chromosome 6B. Four lines, NAU601, NAU615, NAU617, and NAU635, had a part of the short arm of 7Lr#1 transferred to different wheat chromosomes. Four other lines, NAU611, NAU634, NAU633, and NAU618, contained translocations involving Leymus chromosome Lr.7 and different wheat chromosomes. The resistance level of the translocation lines with a single alien chromosome segment was higher than the susceptible wheat parent Chinese Spring but lower than the alien resistant parent L. racemosus. At least three resistance genes in L. racemosus were identified. One was located on chromosome Lr.7, and two could be assigned to the long arm of 5Lr#1 and the short arm of 7Lr#1.     

Transgenic rice plants expressing trichothecene 3-<Emphasis Type="Italic">O</Emphasis>-acetyltransferase show resistance to the <Emphasis Type="Italic">Fusarium</Emphasis> phytotoxin deoxynivalenol

Fusarium head blight (FHB) is a devastating disease of small grain cereal crops caused by the necrotrophic pathogen Fusarium graminearum and Fusarium culmorum. These fungi produce the trichothecene mycotoxin deoxynivalenol (DON) and its derivatives, which enhance the disease development during their interactions with host plants. For the self-protection, the trichothecene producer Fusarium species have Tri101 encoding trichothecene 3-O-acetyltransferase. Although transgenic expression of Tri101 significantly reduced inhibitory action of DON on tobacco plants, there are several conflicting observations regarding the phytotoxicity of 3-acetyldeoxynivalenol (3-ADON) to cereal plants; 3-ADON was reported to be highly phytotoxic to wheat at low concentrations. To examine whether cereal plants show sufficient resistance to 3-ADON, we generated transgenic rice plants with stable expression and inheritance of Tri101. While root growth of wild-type rice plants was severely inhibited by DON in the medium, this fungal toxin was not phytotoxic to the transgenic lines that showed trichothecene 3-O-acetylation activity. This is the first report demonstrating the DON acetylase activity and DON-resistant phenotype of cereal plants expressing the fungal gene. S. Ohsato and T. Ochiai-Fukuda should be considered as joint first authors.     

Minor Differences in Anther Extrusion Affect Resistance to Fusarium Head Blight in Wheat

Fusarium head blight (FHB) remains a serious problem due to yield loss and mycotoxin accumulation in wheat production worldwide. We previously reported that the closed‐flowering (no anther extrusion) characteristic was effective for increasing resistance to FHB infection. In this study, we investigated the relationships between the degree of anther extrusion (AE) and FHB damage using double haploid lines (DHLs), derived from F₁ plants from crosses between closed‐flowering and opened‐flowering varieties. These DHLs exhibited various degrees of AE, and the degree of AE was significantly different among DHLs, regardless of the year and environment (pot‐ or field‐grown). FHB severity was the lowest in closed‐flowering DHLs, and DHLs with partially extruded anthers showed significantly higher FHB symptoms than those with closed‐flowering phenotypes. In general, DHLs with partially extruded anthers also had relatively severe FHB symptoms compared with those exhibiting full anther extrusion. FHB severity was significantly correlated with Fusarium‐damaged kernels and deoxynivalenol concentration. The results of this study showed that partially extruded anthers were considered to be a source of FHB infection. The closed‐flowering phenotype improved resistance to FHB infection. Meanwhile, phenotypes with rapid anther extrusion and ejection also could contribute to the avoidance of FHB infection.     

Durable field resistance to wheat yellow mosaic virus in transgenic wheat containing the antisense virus polymerase gene

Wheat yellow mosaic virus (WYMV) has spread rapidly and causes serious yield losses in the major wheat‐growing areas in China. Because it is vectored by the fungus‐like organism Polymyxa graminis that survives for long periods in soil, it is difficult to eliminate by conventional crop management or fungicides. There is also only limited resistance in commercial cultivars. In this research, fourteen independent transgenic events were obtained by co‐transformation with the antisense NIb8 gene (the NIb replicase of WYMV) and a selectable gene bar. Four original transgenic lines (N12, N13, N14 and N15) and an offspring line (N12‐1) showed high and durable resistance to WYMV in the field. Four resistant lines were shown to have segregated and only contain NIb8 (without bar) by PCR and herbicide resistance testing in the later generations. Line N12‐1 showed broad‐spectrum resistance to WYMV isolates from different sites in China. After growing in the infested soil, WYMV could not be detected by tissue printing and Western blot assays of transgenic wheat. The grain yield of transgenic wheat was about 10% greater than the wild‐type susceptible control. Northern blot and small RNA deep sequencing analyses showed that there was no accumulation of small interfering RNAs targeting the NIb8 gene in transgenic wheat plants, suggesting that transgene RNA silencing, a common mechanism of virus‐derived disease resistance, is not involved in the process of WYMV resistance. This durable and broad‐spectrum resistance to WYMV in transgenic wheat will be useful for alleviating the damage caused by WYMV.