R.J. Zeyen

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.

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.

Hypersensitive cell death, autofluorescence, and insoluble silicon accumulation in barley leaf epidermal cells under attack by Erysiphe graminis f. sp. hordei***

Hypersensitive cell death (HR) of adaxial leaf epidermal cells of barley containing the M1a gene for resistance to powdery mildew germlings, Erysiphe graminis f. sp. hordei , was investigated for temporal relations between cell death, onset of autofluorescence, and accumulation of insoluble silicon (Si). Physiological cell viability or death of barley epidermal cells was determined by concomitant uptake of the vital dye neutral red, and by cell plasmolysis using alpha methyl- d -glucose. Cell death, as judged by lack of neutral red uptake and lack of plasmolysis, first occurred in a low percentage of attacked epidermal cells at 15 h after inoculation and increased to a maximum of 72% at 21 and 24 h. Cells judged dead by physiological criteria always exhibited whole-cell autofluoresence by blue light excitation. Autofluorescence was weak in dying and recently dead cells, but it increased in intensity with time following cell death. It was concluded that autofluorescence accompanies cell death and is perhaps due to release of phenolic compounds from cell vacuoles after cell membranes have lost their semipermeable properties. Insoluble Si accumulation, as determined by energy dispersive X-ray microanalysis using a scanning electron microscope, did not directly correspond to the initial appearance of autofluorescence in HR-dead cells, but occurred gradually after death and in apparent relation to increasing autofluorescence intensity. An hypothesis is stated that phenolics are released by decompartmentalization following cell death and accumulate in the dead cells wall area causing autofluorescence, and that these phenolics form insoluble complexes with Si moving apoplastically in epidermal wall areas due to passive, transpiration stream transport.

The relationship between insoluble silicon and success or failure of attempted primary penetration by powdery mildew (Erysiphe graminis) germlings on barley

Abstract Insoluble silicon (Si) accumulation was quantified in the fungal structures of genetically compatible Erysiphe graminis hordei germlings attempting primary penetration of attached and detached barley leaves and in the leaf cells around the contact sites. This was achieved using previously published procedures of sequential light microscopy, scanning electron microscopy and energy dispersive X-ray microanalysis. Fungal germlings and host cells were sampled and observed at 12, 16, 20 and 24 h after inoculation and the germlings were classified into three categories, viz. successful penetrations, failed penetrations, or indeterminates. With time, relative levels of Si increased in parasite contact regions of host cells, and subsequently in parasite cells, indicating localized accumulation of Si in host cells and Si uptake from host cells into germlings. At 20 h when definite penetration successes and failures by germlings could first be discriminated, far higher Si levels were found in host cell contact sites associated with failed penetration attempts. By 24 h the difference in host cell Si levels between sites of failed and successful penetrations was obscured by continued Si accumulation into haustorial neck and collar regions subtending host cell papilla responses. Thus, the quantity and timing of Si accumulation in host cell regions under attack, is critical to the relationship between Si deposition and failure of fungal germlings to penetrate.

Inhibition of phenylalanine ammonia lyase and cinnamyl alcohol dehydrogenase increases quantitative susceptibility of barley to powdery mildew (Erysiphe graminis D.C.)

Seedling leaves of two barley lines, Algerian/4 * (f14) Man (S) and RISO 5678-S, were excised and supplied either with water (controls), the phenylalanine ammonia lyase inhibitors AOA ( α -aminooxy acetic acid) or AOPP ( α -aminooxy-β-phenylpropionic acid), or the cinnamyl alcohol dehydrogenase inhibitor OH-PAS ([[(2-hydroxyphenyl)amino]sulphinyl]acetic acid, 1,1-di-methylethyl ester) by immersing their cut ends in solution. After 24 h leaves were inoculated with compatible Erysiphe graminis f.sp. hordei conidia and returned to the test solution for 36 h incubation. Treatment with AOA had no effect on the percentage of conidial germlings which penetrated the barleys successfully (formed haustoria), nor on the occurrence of localized autofluorescent host cell responses associated with fungal primary germ-tubes or appressoria. However, both AOPP and OH-PAS treatment led to increased quantitative susceptibility of both barley lines as the percentage of appressoria penetrating host cells to form haustoria was approximately doubled. This was associated with reduction in both the frequency and intensity of autoflu orescent responses associated with fungal germ-tubes; in this respect, the effects of AOPP were slightly more pronounced than those of OH-PAS. The results support the view that in barley, as in oats, autofluorogenic compounds accumulating at sites of germ-tube contact with host cells are phenolic compound(s) synthesized de novo following pathogen attack, that these compound(s) are products synthesized as part of the lignin biosynthetic pathway, and that the compound(s) are involved with resistance to attempted penetration from appressoria.

Effects of two PAL inhibitors on the susceptibility and localized autofluorescent host cell responses of oat leaves attacked by Erysiphe graminis DC

Abstract Seedling leaves of oat (Avena sativa L.) cv. Maldwyn, with adult plant resistance to Erysiphe graminis DC f.sp. avenae ex Merat., were detached and treated with competitive inhibitors of phenylalanine ammonia-lyase (PAL), the enzyme catalysing the first committed step of phenylpropanoid metabolism. The two inhibitors used were α-aminooxy-β-phenylpropionic acid (AOPP) and α-aminooxy acetic acid (AOA). Both chemicals inhibited PAL extracted from seedling leaves, but AOPP was more effective than AOA. To examine effects on host response and pathogen development, cut ends of leaves were immersed in various concentrations of inhibitor for 24 h before inoculation with fungal conidia, and then incubated for a further 36 h. AOPP had no deleterious effects on host tissues or pathogen development, but high (10−3 m ) concentrations of AOA caused loss of leaf turgor, and prevented fungal haustoria from maturing. At high concentrations (⩾ 10−4 m ) both inhibitors increased leaf susceptibility to fungal penetration. Both inhibitors reduced the intensity and frequency of localized autofluorescent host cell responses associated with contact by primary germ tubes and appressorial germ tubes of the fungus. AOPP significantly reduced diameters of autofluorescent host cell responses. The results support the view that the autofluorescent materials are phenolic and that they play a role in the resistance of epidermal cells to penetration. Attempts to reverse the inhibitory effects of AOPP by simultaneous application of cinnamate, the product of PAL activity, were largely unsuccessful.

Effects of the PAL inhibitor, AOPP, on oat, barley and wheat cell responses to appropriate and inappropriate formae specials of Erysiphe graminis DC☆

Abstract Seedling leaves of oat cv. Selma, barley cv. Golden Promise and wheat cv. Cerco, were excised and infused either with water or with 10−3 m α-aminooxy-β-phenylpropionic acid (AOPP), a specific inhibitor of phenylalanine ammonia lyase (PAL). Seedling leaves of the three cereal species from both treatments were inoculated with an isolate of Erysiphe graminis from each of the formae speciales avenae, hordei and tritici. In all appropriate host-parasite combinations, AOPP treatment decreased the percentage of primary germ tubes and appressoria associated with localized autofluorescent host cell responses, and increased the percentage of appressoria forming haustoria i.e. increased the susceptibility of epidermal cells to haustorium formation. Thus, autofluorogens appear to be phenolic compounds associated with resistance to infection of these cereal species by their appropriate forma specialis of E. graminis. AOPP treatment also decreased the percentage of host cells which showed whole-cell autofluorescence, indicating cell death, as a result of attack by an appropriate fungal isolate. In appropriate combinations, whole-cell autofluorescence was most common in the barley f. sp. hordei combination, where decreased epidermal cell death due to AOPP treatment may have contributed to increased rates of haustorium formation. For appropriate combinations this is the first report showing AOPP-associated increase in quantitative susceptibility (reduction in background resistance) for barley and wheat. In inappropriate combinations, AOPP treatment decreased the percentage of primary germ tube contact sites associated with localized autofluorescence, but did not decrease the percentage of appressoria associated with localized autofluorescence. In only one of six inappropriate combinations (oat f. sp. tritici) did AOPP treatment increase rates of haustorium formation above the very low levels seen in all inappropriate host-parasite combinations in water-treated leaves. The failure of AOPP to suppress localized autofluorescent responses to fungal appressoria and to increase susceptibility, in most inappropriate host-parasite combinations suggests that resistance factors operate in inappropriate combinations which are not present in appropriate combinations. Although AOPP treatment decreased the relatively high percentages of dead, autofluorescent cells resulting from inappropriate interactions, decreased cell death was not accompanied by increased haustorium formation i.e. cell susceptibility was not increased.

Suppression of host cinnamyl alcohol dehydrogenase and phenylalanine ammonia lyase increases oat epidermal cell susceptibility to powdery mildew penetration

Seedling leaves of oat cvs Maldwyn and Selma have no known major resistance genes to powdery mildew caused by Erysiphe graminis f.sp. avenae , but their susceptibility to infection is quantitative. Thus, only a portion of fungal germlings successfully overcome cell defences to penetrate host epidermis to form haustoria. OH-PAS ([[(2-hydroxyphenyl)amino] sulphinyl]acetic acid, 1,1-dimethylethyl ester) is a potent, specific suicide inhibitor of CAD (cinnamyl alcohol dehydrogenase), an enzyme specifically involved with synthesis oflignin precursors. OH-PAS was shown to inhibit CAD from oat in vitro . For in vivo assays of effects on epidermal cell defences, the cut ends of excised seedling leaves were immersed in OH-PAS solution for 24 h to allow uptake before inoculation with E. graminis conidia. Inoculated leaves were allowed OH-PAS uptake during a further 36 h incubation period. Initial experiments established that OH-PAS at 10 −3 m decreased the frequency and intensity of localized autofluorescent host epidermal cell responses associated with primary germ-tubes (PGTs) and appressoria. Concurrently, OH-PAS treatment doubled the proportion of appressoria forming haustoria, i.e., it increased quantitative susceptibility by suppressing host cell defences. Similar results were obtained with 10 −3 m AOPP ( α -aminooxy- β -phenyl propionic acid), a competitive inhibitor of PAL (phenylalanine ammonia lyase) which catalyzes the first committed step in phenylpropanoid metabolism. Both inhibitors doubled the proportion of appressoria penetrating epidermal cells and forming haustoria. Both inhibitors reduced the frequency and intensity of localized autofluorescent epidermal host cell responses to PGTs and appressoria, although the effect of AOPP was somewhat greater than that of OH-PAS. Neither OH-PAS nor AOPP had any deleterious effects on fungal development. Results support the idea that host autofluorogens accumulating at sites of fungal germ-tube contact with epidermal cells are phenolic compounds. In addition, the study provides experimental evidence pointing to involvement of products synthesized as part of the lignin biosynthetic pathway in oat epidermal cell defence against attempted penetration by appressoria of E. graminis f.sp. avenae .

Overexpression of the maize Teosinte Branched1 gene in wheat suppresses tiller development

The number of viable shoots influences the overall architecture and productivity of wheat (Triticum aestivum L.). The development of lateral branches, or tillers, largely determines the resultant canopy. Tillers develop from the outgrowth of axillary buds, which form in leaf axils at the crown of the plant. Tiller number can be reduced if axillary buds are not formed or if the outgrowth of these buds is restricted. The teosinte branched1 (tb1) gene in maize, and homologs in rice and Arabidopsis, genetically regulate vegetative branching. In maize, increased expression of the tb1 gene restricts the outgrowth of axillary buds into lateral branches. In this study, the maize tb1 gene was introduced through transformation into the wheat cultivar “Bobwhite” to determine the effect of tb1 overexpression on wheat shoot architecture. Examination of multiple generations of plants reveals that tb1 overexpression in wheat results in reduced tiller and spike number. In addition, the number of spikelets on the spike and leaf number were significantly greater in tb1-expressing plants, and the height of these plants was also reduced. These data reveal that the function of the tb1 gene and genetic regulation of lateral branching via the tb1 mode of action is conserved between wheat, rice, maize and Arabidopsis. Thus, the tb1 gene can be used to alter plant architecture in agriculturally important crops like wheat.

Effects of PAL-specific inhibition on suppression of activated defence and quantitative susceptibility of oats to Erysiphe graminis

Abstract Conidia of Erysiphe graminis DC f.sp. avenae ex. Merat were applied at various densities (low = approximately 30 mm−2, medium = approximately 100 mm−2, high = approximately 300–400 mm−2) to first leaves of spring oat (Avena sativa) cvs Selma and Maldwyn. As previously published, high inoculum density results in activated (enhanced) host cell defence to fungal penetration, and this correlates with elevation in the frequency and intensity of host cell autofluorescent responses. In the current study, leaves were infused with either water (control treatment) or the phenylalanine ammonia lyase (PAL) inhibitors α-aminooxy-β-phenylpropionic acid (AOPP) or α-aminooxy acetic acid (AOA) at concentrations known to be inhibitory to PAL from oats. Results confirm and extend previous observations showing that these PAL inhibitors suppress localized autofluorescent host cell responses to fungal germ tube contact and increase susceptibility to penetration (haustorium formation) from appressoria. The PAL inhibitors, particularly AOPP, suppressed activated defence due to inoculum density and decreased the frequency and intensity of host cell autofluorescence responses. The data support the hypotheses that the activated defence is related to autofluorogenic host responses, and that the autofluorogens are phenolic compounds. Involvement of phenolic compound metabolism in activated defence was shown by assays of the specific activity of PAL from control cv. Selma leaves inoculated with low and high densities of conidia. Compared to uninoculated controls, the specific activity of PAL was enhanced at 6 h after inoculation (when responses to the primary germ tubes of E. graminis were evident), but there was little difference between low and high density inoculations at this time. At 18 h after inoculation (when responses to fungal appressoria were evident), the specific activity of PAL was substantially greater from high than from low inoculum density inoculations. In a coincidental observation, brown colouration (suggesting phenol oxidation) visible in many epidermal cell papillae associated with appressorial attack in control leaves, was not present in most papillae from AOPP-treated leaves.