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.     

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.     

Phytopathogenic fungus Fusarium cerealis in Russia

The fungus Fusarium cerealis is described, which had not been previously identified in Russia. F. cerealis was isolated from wheat and barley roots and grains, as well as from the leaves of thistle collected in the Far Eastern, North Caucasus, and Central regions. F. cerealis strains may be misidentified as F. culmorum or F. graminearum, since the morphological characteristics of these fungi are similar.     

Subcellular targeting of an evolutionarily conserved plant defensin MtDef4.2 determines the outcome of plant–pathogen interaction in transgenic Arabidopsis

The Medicago truncatula gene encoding an evolutionarily conserved antifungal defensin MtDef4.2 was cloned and characterized. In silico expression analysis indicated that MtDef4.2 is expressed in many tissues during the normal growth and development of M. truncatula. MtDef4.2 exhibits potent broad‐spectrum antifungal activity against various Fusarium spp. Transgenic Arabidopsis thaliana lines in which MtDef4.2 was targeted to three different subcellular compartments were generated. These lines were tested for resistance to the obligate biotrophic oomycete Hyaloperonospora arabidopsidis Noco2 and the hemibiotrophic fungal pathogen Fusarium graminearum PH‐1. MtDef4.2 directed to the extracellular space, but not to the vacuole or retained in the endoplasmic reticulum, conferred robust resistance to H. arabidopsidis. Siliques of transgenic Arabidopsis lines expressing either extracellularly or intracellularly targeted MtDef4.2 displayed low levels of resistance to F. graminearum, but accumulated substantially reduced levels of the mycotoxin deoxynivalenol. The data presented here suggest that extracellularly targeted MtDef4.2 is sufficient to provide strong resistance to the biotrophic oomycete, consistent with the extracellular lifestyle of this pathogen. However, the co‐expression of extracellular and intracellular MtDef4.2 is probably required to achieve strong resistance to the hemibiotrophic pathogen F. graminearum which grows extracellularly and intracellularly.     

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.     

Transgenic tobacco and peanut plants expressing a mustard defensin show resistance to fungal pathogens

Defensins are small positively charged, antimicrobial peptides (~5 kDa in size) and some of them exhibit potent antifungal activity. We have cloned the complete cDNA containing an ORF of 243 bp of a defensin of mustard. The deduced amino acid sequence of the peptide showed more than 90% identity to the amino acid sequence of the well-characterized defensins, RsAFP-1 and RsAFP-2 of Raphanus sativus. We have generated and characterized transgenic tobacco and peanut plants constitutively expressing the mustard defensin. Transgenic tobacco plants were resistant to the fungal pathogens, Fusarium moniliforme and Phytophthora parasitica pv. nicotianae. Transgenic peanut plants showed enhanced resistance against the pathogens, Pheaoisariopsis personata and Cercospora arachidicola, which jointly cause serious late leaf spot disease. These observations indicate that the mustard defensin gene can be deployed for deriving fungal disease resistance in transgenic crops.     

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.     

Targeting the pattern-triggered immunity pathway to enhance resistance to Fusarium graminearum

Fusarium head blight (FHB) is a disease of the floral tissues of wheat and barley for which highly resistant varieties are not available. Thus, there is a need to identify genes/mechanisms that can be targeted for the control of this devastating disease. Fusarium graminearum is the primary causal agent of FHB in North America. In addition, it also causes Fusarium seedling blight. Fusarium graminearum can also cause disease in the model plant Arabidopsis thaliana. The Arabidopsis–F. graminearum pathosystem has facilitated the identification of targets for the control of disease caused by this fungus. Here, we show that resistance against F. graminearum can be enhanced by flg22, a bacterial microbe-associated molecular pattern (MAMP). flg22-induced resistance in Arabidopsis requires its cognate pattern recognition receptor (PRR) FLS2, and is accompanied by the up-regulation of WRKY29. The expression of WRKY29, which is associated with pattern-triggered immunity (PTI), is also induced in response to F. graminearum infection. Furthermore, WRKY29 is required for basal resistance as well as flg22-induced resistance to F. graminearum. Moreover, constitutive expression of WRKY29 in Arabidopsis enhances disease resistance. The PTI pathway is also activated in response to F. graminearum infection of wheat. Furthermore, flg22 application and ectopic expression of WRKY29 enhance FHB resistance in wheat. Thus, we conclude that the PTI pathway provides a target for the control of FHB in wheat. We further show that the ectopic expression of WRKY29 in wheat results in shorter stature and early heading time, traits that are important to wheat breeding.     

Resistance against Fusarium Head Blight in Transgenic Wheat Plants Expressing the ScNPR1 gene

Fusarium head blight (FHB) is a severe global wheat disease that may cause severe yield losses, especially during epidemic years. Transforming the regulatory genes in the metabolic pathways of disease resistance into wheat via transgenic methods is one way to improve resistance to FHB. ScNPR1 (Secale cereale‐NPR1), a regulatory gene for systemic acquired resistance (SAR), was isolated from S. cereale cv Jingzhouheimai and transformed into the moderately FHB‐susceptible wheat variety Ningmai 13. RT‐PCR analysis indicated that the ScNPR1 gene was stably expressed in transgenic plants. An evaluation of the resistance to FHB revealed that six ScNPR1 transgenic lines (NP1, NP2, NP3, NP4, NP5 and NP6) exhibited significantly higher FHB resistance than the wild‐type wheat Ningmai 13 and the null‐segregated plants. The expression of pathogenesis‐related (PR) genes after Fusarium graminearum inoculation was earlier or higher than those in the wild‐type variety Ningmai 13. The high expression in the early stages of PR genes should account for the enhanced FHB resistance in the transgenic lines. Our results suggest that overexpression of ScNPR1 could be used to improve FHB resistance in wheat.     

Overexpression of wheat lipid transfer protein gene TaLTP5 increases resistances to Cochliobolus sativus and Fusarium graminearum in transgenic wheat

The fungus Cochliobolus sativus is the main pathogen of common root rot, a serious soil-borne disease of wheat (Triticum aestivum L.). The fungus Fusarium graminearum is the primary pathogen of Fusarium head blight, a devastating disease of wheat worldwide. In this study, the wheat lipid transfer protein gene, TaLTP5, was cloned and evaluated for its ability to suppress disease development in transgenic wheat. TaLTP5 expression was induced after C. sativus infection. The TaLTP5 expression vector, pA25-TaLTP5, was constructed and bombarded into Chinese wheat variety Yangmai 18. Six TaLTP5 transgenic wheat lines were established and characterized. PCR and Southern blot analyses indicated that the introduced TaLTP5 gene was integrated into the genomes of six transgenic wheat lines by distinct patterns, and heritable. RT-PCR and real-time quantitative RT-PCR revealed that the TaLTP5 gene was over-expressed in the transgenic wheat lines compared to segregants lacking the transgene and wild-type wheat plants. Following challenge with C. sativus or F. graminearum, all six transgenic lines overexpressing TaLTP5 exhibited significantly enhanced resistance to both common root rot and Fusarium head blight compared to the untransformed wheat Yangmai 18.     

Antifungal mechanisms of a plant defensin MtDef4 are not conserved between the ascomycete fungi Neurospora crassa and Fusarium graminearum

Defensins play an important role in plant defense against fungal pathogens. The plant defensin, MtDef4, inhibits growth of the ascomycete fungi, Neurospora crassa and Fusarium graminearum, at micromolar concentrations. We have reported that MtDef4 is transported into the cytoplasm of these fungi and exerts its antifungal activity on intracellular targets. Here, we have investigated whether the antifungal mechanisms of MtDef4 are conserved in these fungi. We show that N. crassa and F. graminearum respond differently to MtDef4 challenge. Membrane permeabilization is required for the antifungal activity of MtDef4 against F. graminearum but not against N. crassa. We find that MtDef4 is targeted to different subcellular compartments in each fungus. Internalization of MtDef4 in N. crassa is energy‐dependent and involves endocytosis. By contrast, MtDef4 appears to translocate into F. graminearum autonomously using a partially energy‐dependent pathway. MtDef4 has been shown to bind to the phospholipid phosphatidic acid (PA). We provide evidence that the plasma membrane localized phospholipase D, involved in the biosynthesis of PA, is needed for entry of this defensin in N. crassa, but not in F. graminearum. To our knowledge, this is the first example of a defensin which inhibits the growth of two ascomycete fungi via different mechanisms.     

Constitutive expression of a fusion gene comprising Trigonella foenum-graecum defensin (Tfgd2) and Raphanus sativus antifungal protein (RsAFP2) confers enhanced disease and insect resistance in transgenic tobacco

Plant defensins are small, basic cysteine-rich peptides that can inhibit the growth of a broad range of fungi or bacteria at micro-molar concentrations. They have been introduced as transgenes into different species to enhance host resistance to pathogens. In this study, a fusion gene of two defensins, Trigonella foenum-graecum defensin 2 (Tfgd2) and Raphanus sativus antifungal protein 2 (RsAFP2) fused by a linker peptide of a polyprotein precursor from Impatiens balsamina was introduced into tobacco (Nicotiana tabacum var. Xanthi) via Agrobacterium-mediated leaf section transformation. Putative transgenic plants were confirmed by PCR analysis and integration of the fusion gene was confirmed by Southern blotting. RT-PCR analysis showed that the fusion gene was expressed in several confirmed transgenic plants. Western blotting analysis of crude protein extracts from leaves of the transgenic plants with anti-Tfgd2 and anti-RsAFP2 antibodies exhibited an 8 and 9 kDa bands corresponding to size of the fusion gene and confirmed the expression of fusion protein. When the leaves of transgenic plants were challenged with Rhizoctonia solani and Phytophthora parasitica var. nicotianae pathogens, they showed enhanced levels of disease resistance along with resistance to the generalist herbivore, Spodoptera litura larvae compared to control. Our results demonstrate that Tfgd2–RsAFP2 fusion protein is effective in protecting the transgenic plants against fungal and insect pathogens.     

Pathogenic Variation of Fusarium Isolates Associated with Head Blight of Wheat in Australia

This paper examines the level of pathogenic diversity in Australian Fusarium pseudograminearum and Fusarium graminearum isolates for head blight from the assessment of 51 wheat germplasm lines, barley, triticale, rye, maize and sorghum plants. A set of nine putative wheat differentials were selected and assessed with 10 F. graminearum and 12 F. pseudograminearum isolates. Isolates of both species were pathogenic on all the wheat germplasm lines, barley triticale and rye. The isolates differed largely in a quantitative way with only small differential effects and were statistically demarcated into three pathogenicity groups: low, intermediate and high. Such distribution patterns suggest that wheat germplasm lines employ different resistance mechanisms to each group of isolates and the three pathogenicity groups may have different mechanisms controlling pathogenicity. The aggressiveness of F. graminearum and F. pseudograminearum isolates on the wheat germplasm lines were marginally correlated (r = 0.40). Durum wheats were ranked as the most susceptible while Sumai 3, Ituo Komugi, Sotome A, Sotome and Nobeokabouzu komugi were consistently grouped as resistant by both species. These findings reiterate the need to consider pathogen variability in the screening, selection and improvement of resistance to head blight in wheat.     

Identification and characterization of a fusarium head blight resistance gene TaACT in wheat QTL‐2DL

Fusarium head blight (FHB) resistance in wheat is considered to be polygenic in nature. Cell wall fortification is one of the best resistance mechanisms in wheat against Fusarium graminearum which causes FHB. Metabolomics approach in our study led to the identification of a wide array of resistance‐related (RR) metabolites, among which hydroxycinnamic acid amides (HCAAs), such as coumaroylagmatine and coumaroylputrescine, were the highest fold change RR metabolites in the rachis of a resistant near‐isogenic line (NIL‐R) upon F. graminearum infection. Placement of these metabolites in the secondary metabolic pathway led to the identification of a gene encoding agmatine coumaroyl transferase, herein referred to as TaACT, as a candidate gene. Based on wheat survey sequence, TaACT was located within a FHB quantitative trait loci on chromosome 2DL (FHB QTL‐2DL) between the flanking markers WMC245 and GWM608. Phylogenetic analysis suggested that TaACT shared closest phylogenetic relationship with an ACT ortholog in barley. Sequence analysis of TaACT in resistant and susceptible NILs, with contrasting levels of resistance to FHB, led to the identification of several single nucleotide polymorphisms (SNPs) and two inversions that may be important for gene function. Further, a role for TaACT in FHB resistance was functionally validated by virus‐induced gene silencing (VIGS) in wheat NIL‐R and based on complementation studies in Arabidopsis with act mutant background. The disease severity, fungal biomass and RR metabolite analysis confirmed TaACT as an important gene in wheat FHB QTL‐2DL, conferring resistance to F. graminearum.     

Expression of Lignin Biosynthetic Genes in Wheat during Development and upon Infection by Fungal Pathogens

As a major component of the cell wall, lignin plays an important role in plant development and defense response to pathogens, but negatively impacts biomass processability for biofuels. Silencing the target lignin genes for greater biomass processability should not significantly affect plant development and biomass yield but also must not compromise disease resistance. Here, we report experiments to identify a set of lignin genes that may be silenced without compromising disease resistance. We profiled the expression of 32 lignin biosynthetic candidate genes by qRT-PCR in 17 wheat tissues collected at three developmental stages. Twenty-one genes were expressed at a much higher level in stems compared to sheaths and leaf blades. Expression of seven these genes significantly correlated with lignin content. The co-expression patterns indicated that these 21 genes are under strong developmental regulation and may play a role in lignin biosynthesis. Profiling gene expression of same tissues challenged by two fungal pathogens, Fusarium graminearum and Puccina triticina indicated that expression of 17 genes was induced by F. graminearum. Only PAL1, a non-developmental-regulated gene, was induced by P. triticina. Thus, lignin biosynthetic pathway overlaps defense response to F. graminearum. Based on these criteria, 17 genes, F5H1, F5H2, 4CL2, CCR2, COMT1, and COMT2 in particular that do not overlap with disease resistance pathway, may be the targets for downregulation.     

The plant defensin RsAFP2 induces cell wall stress, septin mislocalization and accumulation of ceramides in Candida albicans

The antifungal plant defensin RsAFP2 isolated from radish interacts with fungal glucosylceramides and induces apoptosis in Candida albicans. To further unravel the mechanism of RsAFP2 antifungal action and tolerance mechanisms, we screened a library of 2868 heterozygous C. albicans deletion mutants and identified 30 RsAFP2‐hypersensitive mutants. The most prominent group of RsAFP2 tolerance genes was involved in cell wall integrity and hyphal growth/septin ring formation. Consistent with these genetic data, we demonstrated that RsAFP2 interacts with the cell wall of C. albicans, which also contains glucosylceramides, and activates the cell wall integrity pathway. Moreover, we found that RsAFP2 induces mislocalization of septins and blocks the yeast‐to‐hypha transition in C. albicans. Increased ceramide levels have previously been shown to result in apoptosis and septin mislocalization. Therefore, ceramide levels in C. albicans membranes were analysed following RsAFP2 treatment and, as expected, increased accumulation of phytoC24‐ceramides in membranes of RsAFP2‐treated C. albicans cells was detected. This is the first report on the interaction of a plant defensin with glucosylceramides in the fungal cell wall, causing cell wall stress, and on the effects of a defensin on septin localization and ceramide accumulation.