DEOXYNIVALENOL DETOXIFICATION IN TRANSGENIC DURUM WHEAT CONFERS RESISTANCE TO FUSARIUM HEAD BLIGHT AND CROWN ROT DISEASES REDUCING GRAIN MYCOTOXIN CONTAMINATION

Fusarium diseases, including Fusarium head blight (FHB) and crown rot (FCR), represent major agricultural problems worldwide, causing reducti on of grain yield, quality and food safety. Grain contaminati on by Fusarium mycotoxins, mainly deoxynivalenol (DON), is responsible for health problems in humans and animals. DON acts as virulence factor during pathogenesis and its glycosylati on, performed by UDPglucosyltransferases (UGTs) and resulti ng in DON-3-glucoside (D3G) producti on, has been identi fi ed as the main detoxifi cati on strategy in wheat. In this work, we produced Triti cum durum cv. Svevo transgenic lines consti tuti vely expressing the barley HvUGT13248 gene. In them, DON-detoxifi cati on by UGT was found to confer a broad-spectrum resistance against F. graminearum and F. culmorum, aff ecti ng diff erent plant organs and developmental stages during FHB and FCR. When challenged with F. graminearum, the transgenic plants revealed a signifi cant reducti on (up to 30%) of FHB symptoms, mostly evident during early-mid stages of the infecti on progress. Notably, much higher DON-to-D3G conversion ability and considerable decrease of DON and DON+D3G content in wholemeal fl our of transgenic lines vs. nontransgenic control was observed. The higher effi ciency of D3G conversion since early infecti on stages may have reduced fungal progression and, consequently, DON and D3G contaminati on in kernels. Furthermore, we highlighted for the fi rst ti me the possible involvement of the DON-detoxifying mechanism in limiti ng FCR disease caused by F. culmorum. When challenged with the pathogen at the seedling stage, the HvUGT13248-expressing lines showed signifi cant reducti on (~50%) of FCR symptoms throughout the infecti on as compared to non-transgenic plants. Transgenic seedlings revealed also a bett er root tolerance to DON, which could have contributed to a higher seedling vigor during the infecti on. The concomitant effi cacy of the DON-detoxifi cati on strategy against FHB and FCR represents an att racti ng sustainable approach to pursue in breeding programs targeti ng broad-spectrum Fusarium resistance and hence reducti on of mycotoxin contaminati on of durum wheat-derived products

EQUIPPING DURUM WHEAT WITH A MAJOR QTL FOR RESISTANCE TO FUSARIUM DISEASES TRANSFERRED FROM THINOPYRUM ELONGATUM AND ITS PYRAMIDING WITH VALUABLE GENES FROM TH. PONTICUM

Favoured by climate changes, Fusarium head blight (FHB), a devastati ng disease of small-grain cereals worldwide, is increasingly spreading also in unusual environments where bread wheat (BW) and durum wheat (DW) are largely culti vated. The scarcity of effi cient resistance sources within adapted germplasm is parti cularly alarming for DW, mainly uti lized for human consumpti on, hence at high risk of kernel contaminati on with health-dangerous mycotoxins. To cope with this scenario, we looked outside the wheat primary genepool and recently transferred an excepti onally eff ecti ve FHB resistance QTL (Fhb-7EL) from the 7EL chromosome arm of the wheatgrass Thinopyrum elongatum onto 7DL of BW recombinant lines. The latt er already possessed valuable genes (Lr19, Yp, yield-related QTL) from a Th. ponti cum 7el1L arm segment, distally inserted onto 7DL. BW lines with the 7el1L+7EL assembly were crossed with previously developed DW-Th. ponti cum recombinants, having small 7el1L distal segments on 7AL arms. As proved by GISH analysis, homologous pairing occurred with high frequency in the shared 7el1L region between the BW and DW recombinant chromosomes. As a result, desirable 7EL+7el1L recombinant types with 2n = 28 were isolated in the BC1 progeny to DW, aided by PCR-based markers. Homozygous recombinant plants selected in the BC2F2 progeny were challenged by Fusarium graminearum spike inoculati on to verify the Fhb-7EL QTL effi cacy into the DW background. Infecti on outcomes confi rmed what previously observed in BW, with around 90% reducti on of disease severity vs. suscepti ble controls, invariably associated with presence of the Fhb-7EL QTL. Moreover, as for the BW types, the same Fhb-7EL was found to provide the novel DW recombinants also with resistance to the crown rot disease, as from seedling infecti on with F. culmorum. Through alien segment pyramiding we succeeded in equipping DW with a very eff ecti ve barrier against diff erent Fusarium diseases and other positi ve att ributes contributi ng to yield security and safety

EXPRESSION OF BEAN PGIP2 UNDER CONTROL OF THE BARLEY LEM1 PROMOTER LIMITS FUSARIUM GRAMINEARUM INFECTION IN WHEAT

Fusarium Head Blight (FHB) caused by Fusarium graminearum is one of the most destructive fungal diseases of wheat worldwide. The pathogen infects the spike at flowering time and causes severe yield losses, deterioration of grain quality, and accumulation of mycotoxins. Better understanding of the means of pathogen entry and colonization of floral tissue is crucial to providing effective protection against FHB. Polygalacturonase inhibiting proteins (PGIPs) are cell wall proteins that inhibit the activity of polygalacturonases (PGs), a class of pectin-depolymerizing enzymes secreted by microbial pathogens, including Fusaria. The constitutive expression of a bean PGIP (PvPGIP2) under control of the maize Ubi1 promoter limits FHB symptoms and reduces mycotoxin accumulation in wheat grain [Janni et al. 2008 Molec. Plant Microb. Interact. 21:171]. To better understand which spike tissues play major roles in limiting F. graminearum infection, we explored the use of PvPGIP2 to defend specific spike tissues by expressing it under control of the barley Lem1 promoter [Somleva and Blechl 2005 Cer. Res. Comm. 33:665]. We show here that the expression of PvPGIP2 in lemma, palea, rachis and anthers reduced FHB symptoms caused by F. graminearum compared to symptoms in infected nontransgenic plants. However, the expression of PvPGIP2 only in the endosperm under control of a HMW-glutenin gene promoter did not affect FHB symptom development, indicating that once the pathogen has reached the endosperm, inhibition of the pathogen\u2019s PG activity is not effective in preventing its further spread

The Fusarium graminearum FGSG_03624 xylanase primes plant immune responses and increases plant disease resistance

Plants activate defense responses against pathogens once pattern recognition receptors (PRRs) perceive the presence of pathogen-associated molecular patterns (PAMPs). Some cell wall degradating enzymes function as PAMPs and activate the plant immune responses, as shown for EIX and Xyn11A xylanases of Trichoderma viride and Botrytis cinerea, respectively. Since the Fusarium graminearum FGSG_03624 xylanase has been shown to induce necrosis and hydrogen peroxide accumulation in wheat independently from its enzymatic activity, we investigated its ability to modulate plant immunity. To this aim, we transiently and constitutively expressed an enzymatically inactivated form of FGSG_03624 in tobacco and Arabidopsis, respectively. Afterwards, these plants were challenged with Pseudomonas syringae pv. maculicola and B. cinerea. Effectiveness in reducing symptoms caused by the bacterial pathogen was evident both in Arabidopsis and in tobacco whilst no symptoms reduction was observed after B. cinerea infection. The increased resistance of Arabidopsis plants to P. syringae was associated to a faster and stronger activation of jasmonate/ethylene and salicylate pathways detected after pathogen inoculation. The priming effect was also confirmed after F. graminearum inoculation of durum wheat spikes exogenously treated with the FGSG_03624 xylanase. Indeed, wheat spikes treated with the xylanase exhibited reduced symptoms in the early phases of infection and a lower fungal biomass accumulation in semolina compared to control. Besides, callose deposition was detected in infected spikes previously treated with the xylanase and not in infected control plants. In conclusion, our results highlight the ability of FGSG_03624 to prime defense responses in plants thus conferring disease resistance

Constitutive expression of the xylanase inhibitor TAXI-III delays Fusarium Head Blight symptoms in durum wheat transgenic plants

Cereals contain xylanase inhibitor proteins (XIs) which inhibit microbial xylanases and are considered part of the defence mechanisms to counteract microbial pathogens. Nevertheless, in planta evidences for this role have not been reported yet. Therefore, we produced a number of transgenic plants constitutively over-expressing TAXI-III, a member of the TAXI type XIs that is induced by pathogen infection. Results showed that TAXI-III endows the transgenic wheat with new inhibition capacities. We showed also that TAXI-III is correctly secreted into the apoplast and possesses the expected inhibition parameters against microbial xylanases. The new inhibition properties of the transgenic plants correlate with a significant delay of Fusarium Head Blight (FHB) disease symptoms caused by Fusarium graminearum but do not influence significantly leaf spot symptoms caused by Bipolaris sorokiniana. We showed that this contrasting result can be due to the different capacity of TAXI-III to inhibit the xylanase activity of these two fungal pathogens. These results provide for the first time straight evidence in planta that XIs are involved in plant defence against fungal pathogens and show the potential to manipulate TAXI-III accumulation to improve wheat resistance against F. graminearum