Relationship between the D genome of hexaploid wheats (AABBDD) and Ae. squarrosa as deduced by seed storage proteins and molecular marker analyses

The electrophoretical analyses of seed storage protein components from the gliadin and glutenin fractions in T. aeslivum ssp. vulgare, compaction, sphaerococcum, macha, vavilovii, and spelta have revealed limited variation at the tightly linked coding loci Gli-D1/Glu-D3, and Glu-D1, located respectively on the short and long arm of chromosome ID, and at the GH-D2 locus, positioned on the short arm of chromosome 6D. Much higher variation was observed, for the same protein components, in the wild diploid Ae. squarrosa, the D genome donor of the aestivum group. Genetic variation in the same wheat subspecies and in Ae. squarrosa has also been evaluated by Southern hybridization of genomic DNAs, which were digested with several restriction enzymes, and hybridized with cloned sequences of genes coding for seed storage proteins. The much higher degree of variation observed for the seed storage protein genes of Ae. squarrosa, in comparison with the variation exhibited by the proteins encoded by the D genome chromosomes of hexaploid wheats, supports the hypothesis that a limited number of crosses gave rise to hexaploid wheats of the aestivum group

Transfer of a major qtl for resistance to fusarium head blight from Thinopyrum elongatum onto durum wheat 7al chromosome arm and its pyramiding with other useful genes from Th. ponticum

Fusarium Head Blight (FHB), also called scab, a major wheat disease worldwide, has in recent years become a threat also in uncommon cultivation areas of bread and durum wheat. The lack of efficient and durable sources of resistance within adapted durum wheat germplasm is particularly alarming. In fact, durum wheat is used almost exclusively for human consumption, and Fusarium attacks, besides causing severe yield losses, pose a high risk of kernel contamination with health-dangerous mycotoxins, notably deoxynivalenol (DON). To cope with this scenario, looking outside of the primary genepool and exploring exotic variability harboured within related Triticeae species, appears as a worth strategy. We recently mapped an exceptionally effective QTL for FHB resistance (provisional designation Fhb-7EL) onto the telomeric portion of the 7EL chromosome arm of diploid Thinopyrum elongatum, and pyramided it with other useful genes from the 7el1L arm of decaploid Th. ponticum (Lr19, Yp, yield related traits) onto the 7DL arm of bread wheat- Thinopyrum recombinant lines (Ceoloni et al. TAG, in press). Two such FHB resistant recombinants, having distal 70% of their 7DL replaced by chromatin of the two Thinopyrum species in slightly different relative proportions, were crossed with two previously developed durum wheat- Th. ponticum recombinants, having 23% and 28% of their 7AL arm replaced by 7el1L chromatin, respectively. In the shared 7el1L region between the bread wheat (6x) and durum wheat (4x) parental recombinant types, including in all cases the Lr19 gene, homologous pairing evidently occurred with high frequency (over 70%), as proved by GISH-based analysis of meiotic pairing of 5x F1\u2019s. As a result, new 7EL-7el1L recombinant types could be isolated in the BC1 generation to durum wheat. Selection for desired recombinants, i.e. those involving chromosome 7A and with a total Thinopyrum spp. (7EL+7el1L) chromatin not exceeding the 28% of the arm, was carried out by a panel of user-friendly PCR-based markers. To accelerate reduction of chromosome number to the euploid 2n=28, in vitro culture of BC2 embryos was carried out. Selected heterozygous recombinant plants, most of them having reached a stable chromosomal condition, were selfed and in the BC2F2 progeny homozygous genotypes were isolated. The latter were challenged by Fusarium graminearum spike inoculation, to verify the efficacy of the Fhb-7EL QTL once inserted into a durum wheat background. Compared to highly susceptible control plants, inoculation outcomes confirmed what previously observed at the bread wheat level, with remarkable reduction of disease severity, averaging over 90%, invariably associated with presence of the Fhb-7EL QTL. Thus, what is considered to be by far the most effective resistance to FHB available, is now readily exploitable in breeding to provide protection against this threatening disease, hence substantially enhancing food security and safety of the bread and durum wheat crop, in Italy and worldwide

The expression of a bean PGIP in transgenic wheat confers increased resistance to the fungal pathogen Bipolaris sorokiniana

In several plant-pathogen interactions to overcome the barrier represented by cell wall most fungal pathogens produce a variety of hydrolytic enzymes and between them PGs are one of the first to be secreted. We demonstrate that transgenic wheat plants expressing PvPGIP2 showed a significant reduction of symptoms following the infection of Bipolaris sorokiniana suggesting that pectin hydrolysis is an important step for fungal penetration of wheat plants.In molti sistemi pianta patogeno i patogeni al fine di superare l’ostacolo rappresentato dalla parete cellulare producono degli enzimi idrolitici tra cui le Poligalatturonasi ( PG) sono tra i primi ad essere secreti. In questo lavoro dimostriamo che piante transgeniche di frumento sovraesprimenti la PvPGIP2 mostrano una significativa riduzione nella sintomatologia riscontrata in seguito ad infezione con Bipolaris sorokiniana suggerendo che l’idrolisi della pectina rappresenta uno step importante per la penetrazione e la colonizzazione dei tessuti di frumento.L'articolo é disponibile sul sito dell'editore: http://www.apsjournals.apsnet.or

Reinforcing and broadening resistance against Fusarium diseases in durum wheat by an udp-glucosyltransferase transgene and its pyramiding with a pectin methyl esterase inhibitor transgene

Many species of the genus Fusarium are phytopathogenic fungi of a wide range of cereal crop plants, including wheat. F. graminearum is the main causal agent of Fusarium Head Blight (FHB), while F. culmorum and F. pseudograminearum are the main responsible species of Fusarium Crown Rot (FCR). Fusarium diseases represent major agricultural problems worldwide, causing reduction of grain yield, grain quality and food safety. The latter is associated with contamination of grains with mycotoxins, particularly deoxynivalenol (DON), which cause health problems in humans and animals. DON is a protein synthesis inhibitor, acting as a virulence factor during pathogenesis and resulting essential for fungal spread along the spike. Conversion of DON to deoxynivalenol-3-\u3b2-D-glucoside (D3G) by the activity of specific UDP-glucosyltransferases (UGTs), is one of the mechanisms involved in enhancing plant tolerance to DON. Previous studies demonstrated that the expression of the barley HvUGT13248 gene confers resistance to DON in Arabidopsis thaliana (Shin et al. 2012, J Exp Bot. 63:4731-40) and type II resistance to FHB (i.e. resistance to fungal spread within host tissues) in bread wheat (Li et al. 2015, MPMI 28:1237-46). Improvement of FHB resistance is a major target in both bread and durum wheat. The latter, however, is especially vulnerable, as effective sources of resistance are particularly limited. Therefore, we decided to verify whether the expression of the HvUGT13248 gene could enhance FHB resistance in durum wheat as well. To this aim, transgenic lines of Triticum durum cv. Svevo, constitutively expressing the HvUGT13248 gene, were produced. Transgenic plants in which presence of transcript and protein was confirmed, were infected with F. graminearum and evaluated for FHB severity, DON content and D3G conversion as compared to wild type plants. Our results showed that the HvUGT13248 gene determines in durum wheat a significant reduction of FHB symptoms (up to 30%) compared to control plants. This effect, however, was mainly evident at early infection stages, progressively decreasing at later stages. This outcome differs from what observed in transgenic bread wheat expressing the same UGT gene, in which FHB severity did not exceed 20% up to the last stages of infection (Li et al. 2015). To verify further the effectiveness of the DON-detoxifying approach, durum wheat lines with the same HvUGT13248 transgene were challenged with F. culmorum, also able to produce DON. A significant reduction of FCR symptoms compared to Svevo plants was observed. This represents the first report of improvement of FCR resistance associated with overexpression of an UGT involved in DON-detoxification. Recently, in order to combine in the same plant genes controlling two different mechanisms of type II resistance to FHB, we have crossed two types of durum wheat transgenic lines, one expressing the HvUGT13248 gene, the other AcPMEI, coding for a kiwi pectin methyl esterase inhibitor, known to increase resistance by strengthening the cell wall pectin fraction. On selected carriers of both transgenes, and in control lines with individual or no transgenes, the efficacy of the novel assembly will be verified against FHB and FCR

Tissue-specific expression of PvPGIP2 to improve wheat resistance against Fusarium graminearum

Fusarium Head Blight (FHB) is one of the most important wheat diseases caused by some fungi of the genus Fusarium. The pathogen infects the spike at flowering time and causes severe yield losses and deterioration of grain quality due to the secretion of mycotoxins during infection. The understanding of the precise mode of pathogen entering and the subsequent floral tissue colonize is a crucial point to control FHB. Polygalacturonase inhibiting proteins (PGIPs) are cell wall proteins that inhibit the pectin-depolymerizing activity of polygalacturonases (PGs) secreted by microbial pathogens and insects. The constitutive expression of the bean PvPGIP2 limits FHB symptoms and reduces mycotoxin accumulation in wheat. To better understand the spike tissues that play a role in limiting Fusarium infection, we have produced transgenic wheat plants expressing PvPGIP2 or in the endosperm or simultaneously in lemma, palea, anthers and rachis. We showed that this latter approach reduced FHB symptoms caused by F. graminearum compared to control non transgenic plants. The extent of disease symptom reduction was similar to what obtained when PvPGIP2 was expressed constitutively. We showed also that different level of PvPGIP2 accumulation produced similar level of protection. Conversely, the expression of PvPGIP2 only in the endosperm did not affect FHB symptom development, indicating that when the pathogen has reached the endosperm, inhibition of the polygalacturonase (PG) activity of the pathogen is ineffective to prevent fungal spread. Probably the rich source of the endosperm tissue makes the PG activity dispensable for pathogen colonization. Alternatively, the massive growth of the fungus at this stage produces a large amount of PG that is not inhibited by the available PGIP

Production and characterization of wheat lines silenced in alpha amylase/trypsin inhibitor genes involved in adverse reactions to wheat

Although wheat is the most consumed crop worldwide, it is also the main factor triggering different adverse reactions, among which celiac disease, true allergies and Non Celiac Wheat Sensitivity (NCWS). Among allergies, the so called \u201cbaker\u2019s asthma\u201d, is the most common professional asthma in Europe and is caused mainly by proteins present in the soluble fraction, especially alpha-amylase/trypsin inhibitors (ATI). Recent findings indicate in this class of proteins also the main factor triggering NCWS, that at present affects people with a frequency around 1:80, higher than celiac disease (1:100), but this is still a matter of debate. On this basis, we have produced RNAi wheat plants (both durum and bread wheat) in which different ATI genes have been silenced, to be used as a proof of concept, in order to test if they have a minor impact on adverse reactions, by using in vitro tests. We have silenced CM3, CM16 and 0.28 genes and have now available several lines in T4 generation. ELISA tests and immunoblotting analysis, by using a monoclonal antibody against ATI proteins, have shown that RNAi silenced wheat kernels present a lower amount of ATI proteins. Moreover, we are characterizing these lines in relation to respiratory allergies. Protein extracts from silenced plants are being tested by using human sera of allergic patients in order to verify if a lower amount of immunogenic polypeptides is recognized in comparison to wild type untransformed plants. If this is the case, the realization of new wheat genotypes expressing a lower amount of ATI proteins can be a realistic target to be reached by classical breeding procedures

A major qtl for resistance to fusarium head blight and Crown rot of wheat on Thinopyrum elongatum chromosome 7e: cytogenetic mapping and assembling into bread wheat with valuable genes from Th. Ponticum

Among wheat relatives, the Thinopyrum genus represents one of the richest sources of valuable genes/QTL for wheat improvement. One notable and still unexploited trait is the exceptionally effective resistance to Fusarium Head Blight (FHB) originating from a diploid member of the genus, Thinopyrum elongatum. Up to date, the resistance was only associated to the long arm of Th. elongatum chromosome 7E (7EL), while detailed genetic mapping of the responsible gene/QTL was still lacking. We targeted the transfer of the temporarily designated Fhb- 7EL locus into bread wheat, by pyramiding it with other valuable genes/QTL (Lr19 for leaf rust resistance, yield-related traits) and included in a Th. ponticum 7el1L segment, stably inserted into the wheat 7DL arm of line T4. Mapping of the Fhb-7EL QTL was here based on a bioassay with Fusarium graminearum, the main causal agent of FHB, of different 7EL-7el1L bread wheat recombinant lines. Nine such recombinant types were successfully obtained without resorting to any genetic pairing promotion, but relying on the 7EL-7el1L close homoeology. Pairing between the two critical arms was in fact observed by Genomic In Situ Hybridization (GISH) at meiotic metaphase I of F1 plants between the 7E(7D) substitution line and the translocation line T4 (70% distal 7el1L on 7DL), which resulted in 14% 7EL-7el1L recombination frequency. The Fhb-7EL locus was mapped to the telomeric portion of 7EL, associated with marker loci XBE405003 Xsdauk66 and Xcfa2240. FHB resistant recombinants, with useful combinations of more proximally located 7el1L genes/QTL, could be selected. The transferred Fhb-7EL locus was shown to reduce disease severity at the spike level and fungal biomass in the grains of infected recombinants by over 95%. The same Fhb-7EL QTL was, for the first time, proved to be effective also against F. culmorum and F. pseudograminearum, predominant agents of Fusarium Crown Rot (FCR). Yield performance in preliminary field tests of the pre-breeding lines possessing a suitable 7EL-7el1L gene/QTL assembly showed to be very promising. Given the expected inheritance as a unit of the composite Thinopyrum segment in cross progeny with wheat, either of the several co-dominant PCR-based markers identified in the course of the work will enable easy tracking of the novel gene/QTL assembly in transfer programs into adapted cultivars

Characterization of the complex locus of bean encoding polygalacturonase-inhibiting proteins reveals subfunctionalization for defense against fungi and insects.

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant inhibitors of fungal endopolygalacturonases (PGs) that belong to the superfamily of Leu-rich repeat proteins. We have characterized the full complement of pgip genes in the bean (Phaseolus vulgaris) genotype BAT93. This comprises four clustered members that span a 50-kb region and, based on their similarity, form two pairs (Pvpgip1/Pvpgip2 and Pvpgip3/Pvpgip4). Characterization of the encoded products revealed both partial redundancy and subfunctionalization against fungal-derived PGs. Notably, the pair PvPGIP3/PvPGIP4 also inhibited PGs of two mirid bugs (Lygus rugulipennis and Adelphocoris lineolatus). Characterization of Pvpgip genes of Pinto bean showed variations limited to single synonymous substitutions or small deletions. A three-amino acid deletion encompassing a residue previously identified as crucial for recognition of PG of Fusarium moniliforme was responsible for the inability of BAT93 PvPGIP2 to inhibit this enzyme. Consistent with the large variations observed in the promoter sequences, reverse transcription-PCR expression analysis revealed that the different family members differentially respond to elicitors, wounding, and salicylic acid. We conclude that both biochemical and regulatory redundancy and subfunctionalization of pgip genes are important for the adaptation of plants to pathogenic fungi and phytophagous insects