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 Fusarium spp.. 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 pathogens. The constitutive expression of the bean PvPGIP2 limits FHB symptoms and reduces mycotoxin accumulation in wheat. To better understand which spike tissues plays a role in limiting Fusarium infection, we have produced transgenic wheat plants expressing PvPGIP2 in the endosperm or simultaneously in lemma, palea, anthers and rachis. This latter approach reduced FHB symptoms, whereas the expression of PvPGIP2 only in the endosperm did not affect FHB development, indicating that when the pathogen has reached the endosperm, inhibition of pathogen PGs ineffective to prevent fungal spread

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

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

Cytogenetic mapping of a major locus for resistance to Fusarium head blight and crown rot of wheat on Thinopyrum elongatum 7EL and its pyramiding with valuable genes from a Th. ponticum homoeologous arm onto bread wheat 7DL

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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

Enhancing grain size in durum wheat using RNAi to knockdown GW2 genes

Sestili F., Pagliarello R., Zega A., Saletti R., Pucci A., Botticella E., Masci S., Tundo S., Moscetti I., Foti S., Lafiandra D. 2019 Enhancing grain size in durum wheat using RNAi to knock-down GW2 genes. Theoretical and Applied Genetics, 132(2): 419-429 https://doi.org/10.1007/s00122-018-3229-9. Abstract Key message Knocking down GW2 enhances grain size by regulating genes encoding the synthesis of cytokinin, gibberellin, starch and cell wall. Abstract Raising crop yield is a priority task in the light of the continuing growth of the world’s population and the inexorable loss of arable land to urbanization. Here, the RNAi approach was taken to reduce the abundance of Grain Weight 2 (GW2) transcript in the durum wheat cultivar Svevo. The effect of the knockdown was to increase the grains’ starch content by 10–40%, their width by 4–13% and their surface area by 3–5%. Transcriptomic profiling, based on a quantitative real-time PCR platform, revealed that the transcript abundance of genes encoding both cytokinin dehydrogenase 1 and the large subunit of ADP-glucose pyrophosphorylase was markedly increased in the transgenic lines, whereas that of the genes encoding cytokinin dehydrogenase 2 and gibberellin 3-oxidase was reduced. A proteomic analysis of the non-storage fraction extracted from mature grains detected that eleven proteins were differentially represented in the transgenic compared to wild-type grain: some of these were involved, or at least potentially involved, in cell wall development, suggesting a role of GW2 in the regulation of cell division in the wheat grain

Interaction of human hemoglobin and semi-hemoglobins with the Staphylococcus aureus hemophore IsdB: a kinetic and mechanistic insight

Among multidrug-resistant bacteria, methicillin-resistant Staphylococcus aureus is emerging as one of the most threatening pathogens. S. aureus exploits different mechanisms for its iron supply, but the preferred one is acquisition of organic iron through the expression of hemoglobin (Hb) receptors. One of these, IsdB, belonging to the Isd (Iron-Regulated Surface Determinant) system, was shown to be essential for bacterial growth and virulence. Therefore, interaction of IsdB with Hb represents a promising target for the rational design of a new class of antibacterial molecules. However, despite recent investigations, many structural and mechanistic details of complex formation and heme extraction process are still elusive. By combining site-directed mutagenesis, absorption spectroscopy, surface plasmon resonance and molecular dynamics simulations, we tackled most of the so far unanswered questions: (i) the exact complex stoichiometry, (ii) the microscopic kinetic rates of complex formation, (iii) the IsdB selectivity for binding to, and extracting heme from, α and β subunits of Hb, iv) the role of specific amino acid residues and structural regions in driving complex formation and heme transfer, and (v) the structural/dynamic effect played by the hemophore on Hb

A retrospective multicentric observational study of trastuzumab emtansine in HER2 positive metastatic breast cancer: A real-world experience

We addressed trastuzumab emtansine (T-DM1) efficacy in HER2+ metastatic breast cancer patients treated in real-world practice, and its activity in pertuzumab-pretreated patients. We conducted a retrospective, observational study involving 23 cancer centres, and 250 patients. Survival data were analyzed by Kaplan Meier curves and log rank test. Factors testing significant in univariate analysis were tested in multivariate models. Median follow-up was 15 months and median T-DM1 treatment-length 4 months. Response rate was 41.6%, clinical benefit 60.9%. Median progression-free and median overall survival were 6 and 20 months, respectively. Overall, no differences emerged by pertuzumab pretreatment, with median progression-free and median overall survival of 4 and 17 months in pertuzumab-pretreated (p=0.13), and 6 and 22 months in pertuzumab-na\uc3\uafve patients (p=0.27). Patients who received second-line T-DM1 had median progression-free and median overall survival of 3 and 12 months (p=0.0001) if pertuzumab-pretreated, and 8 and 26 months if pertuzumab-na\uc3\uafve (p=0.06). In contrast, in third-line and beyond, median progression-free and median overall survival were 16 and 18 months in pertuzumab-pretreated (p=0.05) and 6 and 17 months in pertuzumab-na\uc3\uafve patients (p=0.30). In multivariate analysis, lower ECOG performance status was associated with progression-free survival benefit (p < 0.0001), while overall survival was positively affected by lower ECOG PS (p < 0.0001), absence of brain metastases (p 0.05), and clinical benefit (p < 0.0001). Our results are comparable with those from randomized trials. Further studies are warranted to confirm and interpret our data on apparently lower T-DM1 efficacy when given as second-line treatment after pertuzumab, and on the optimal sequence order