Two pathogenesis-related proteins interact with leucine-rich repeat proteins to promote Alternaria leaf spot resistance in apple

Alternaria leaf spot in apple (Malus x domestica), caused by the fungal pathogen Alternaria alternata f. sp. mali (also called A. mali), is a devastating disease resulting in substantial economic losses. We previously established that the resistance (R) protein MdRNL2, containing a coiled-coil, nucleotide-binding, and leucine-rich repeat (CCR-NB-LRR) domain, interacts with another CCR-NB-LRR protein, MdRNL6, to form a MdRNL2–MdRNL6 complex that confers resistance to A. mali. Here, to investigate the function of the MdRNL2–MdRNL6 complex, we identified two novel pathogenesis-related (PR) proteins, MdPR10-1 and MdPR10-2, that interact with MdRNL2. Yeast two-hybrid (Y2H) assays and bimolecular fluorescence complementation (BiFC) assays confirmed that MdPR10-1 and MdPR10-2 interact with MdRNL2 and MdRNL6 at the leucine-rich repeat domain. Transient expression assays demonstrated that accumulation of MdPR10-1 and MdPR10-2 enhanced the resistance of apple to four strains of A. mali that we tested: ALT1, GBYB2, BXSB5, and BXSB7. In vitro antifungal activity assays demonstrated that both the proteins contribute to Alternaria leaf spot resistance by inhibiting fungal growth. Our data provide evidence for a novel regulatory mechanism in which MdRNL2 and MdRNL6 interact with MdPR10-1 and MdPR10-2 to inhibit fungal growth, thereby contributing to Alternaria leaf spot resistance in apple. The identification of these two novel PR proteins will facilitate breeding for fungal disease resistance in apple.

Study of Triticum aestivum Resistome in Response to Wheat dwarf India Virus Infection

Susceptible and resistant germplasm respond differently to pathogenic attack, including virus infections. We compared the transcriptome changes between a resistant wheat cultivar, Sonalika, and a susceptible cultivar, WL711, to understand this process in wheat against wheat dwarf India virus (WDIV) infection. A total of 2760 and 1853 genes were differentially expressed in virus-infected and mock-inoculated Sonalika, respectively, compared to WL711. The overrepresentation of genes involved in signaling, hormone metabolism, enzymes, secondary metabolites, proteolysis, and transcription factors was documented, including the overexpression of multiple PR proteins. We hypothesize that the virus resistance in Sonalika is likely due to strong intracellular surveillance via the action of multiple PR proteins (PR1, RAR1, and RPM1) and ChiB. Other genes such as PIP1, LIP1, DnaJ, defensins, oxalate oxidase, ankyrin repeat protein, serine-threonine kinase, SR proteins, beta-1,3-glucanases, and O-methyltransferases had a significant differential expression and play roles in stress tolerance, may also be contributing towards the virus resistance in Sonalika. In addition, we identified putative genes with unknown functions, which are only expressed in response to WDIV infection in Sonalika. The role of these genes could be further validated and utilized in engineering resistance in wheat and other crops.

Χαρακτηρισμός του Bacillus subtilis MBI600 ως παράγοντα βιολογικής καταπολέμησης φυτοπαθογόνων και επαγωγής της ανάπτυξης φυτών

Η χρήση ωφέλιμων μικροοργανισμών στην σύγχρονη γεωργία δεν αποτελεί πλέον εξαίρεση αλλά είναι απαραίτητη σε όλες τις καλλιεργητικές στρατηγικές. Τα ωφέλιμα ριζοβακτήρια αποτελούν ένα σημαντικό μέρος των εμπορικών σκευασμάτων, εξαιτίας της ικανότητας τους να προωθούν την ανάπτυξη των φυτών και να είναι αποτελεσματικά έναντι παθογόνων μικροοργανισμών. Βακτήρια που ανήκουν στο γένος Bacillus, αποτελούν την πλειοψηφία των μικροοργανισμών που διατίθενται εμπορικά ως παράγοντες βιολογικής καταπολέμησης ασθενειών των φυτών. Το εμπορικό σκεύασμα Serifel® , εμπεριέχει τον βιολογικό παράγοντα BacillussubtilisMBI600 (BsMBI600), και στην παρούσα διατριβή παρουσιάζεται μια συστηματική καταγραφή των μηχανισμών δράσης του συγκεκριμένου ωφέλιμου μικροοργανισμού. Για την καλύτερη κατανόηση των μηχανισμών δράσης του BsMBI600, πραγματοποιήθηκε αλληλούχιση του γονιδιώματος του μικροοργανισμού με Illumina HiSeq X. Τα αποτελέσματα της ανάλυσης ανέδειξαν γονίδια που σχετίζονται με την παραγωγή ουσιών που δρουν ως σήματα επικοινωνίας με τα φυτά-ξενιστές, οδηγώντας σε επιτυχημένο αποικισμό των ριζών. Επιπλέον, ταυτοποιήθηκαν γονίδια που σχετίζονται με την ικανότητα των βιολογικών παραγόντων να αυξάνουν την διαθεσιμότητα των θρεπτικών στοιχείων των φυτών, αλλά και την παραγωγή φυτοορμονών που οδηγούν στην προαγωγή της ανάπτυξης των φυτών. Τέλος, ταυτοποιήθηκαν γονίδια που σχετίζονται με την παραγωγή αντιμικροβιακών ουσιών, στο γονιδίωμα το BsMBI600.H ικανότητα αποικισμού του BsMBI600 σε ρίζες φυτών τομάτας και αγγουριάς, μελετήθηκε σε διαφορετικά υποστρώματα ανάπτυξης. Βακτηριακά κύτταρά του στελέχους, μετασχηματίσθηκαν με την εισαγωγή μια κίτρινης φθορίζουσας πρωτεΐνης, με τον τρόπο της φυσικής ιδιότητας των βακτηρίων να επιτρέπουν την εισαγωγή ξένων μορίων μέσα στο κύτταρο τους (naturaltransformation). Τα μετασχηματισμένα στελέχη του BsMBI600, μελετήθηκαν με την χρήση ανεστραμμένου φθορίζοντος μικροσκοπίου, και καταγράφηκε ο επιφυτικός αποικισμός 48 ώρες μετά την εφαρμογή. Εν συνεχεία, καταγράφθηκε η ικανότητα αποικισμού σε διαφορετικά υποστρώματα ανάπτυξης (κύβοι υδροπονίας, 2 τύποι εδαφών και αποστειρωμένο σύστημα). Ο βιολογικός παράγοντας BsMBI600, κατάφερε να αποικίσει το ριζικό σύστημα των 2 ξενιστών σε όλα τα υποστρώματα, με μεγάλες διακυμάνσεις στην διάρκεια και στην επιβίωση βακτηριακών κυττάρων, οδηγώντας στο συμπέρασμα, πως το εδαφικό υπόστρωμα παίζει σημαντικό ρόλο στην ικανότητα αποικισμού.Η ικανότητα του BsMBI 600 να προάγει την ανάπτυξη των φυτών αλλά και να ελέγχει σημαντικά παθογόνα των 2 ξενιστών, επιβεβαιώθηκε από ένα σύνολο πειραμάτων που πραγματοποιήθηκαν. Εφαρμογές με τον βιολογικό παράγοντα, οδήγησαν σε φυτά που παρουσίασαν αύξηση σε διάφορους παράγοντες (μήκος στελέχους και ρίζας, φωτοσύνθεση κ.α.). Η βιολογική δράση του έναντι φυτοπαθογόνων μικροοργανισμών επιβεβαιώθηκε τόσο με invitro πειράματα, όσο και με πειράματα σε φυτοδοχεία. Ο BsMBI600 μείωσε την εμφάνιση και την ένταση σημαντικών ασθενειών που προκαλούνται από τα παθογόνα Rhizoctonia solani, Pythium ultimum, P. Aphanidernatum,Fusarium oxysporum f.sp. radicis-lycopersici -Forl, F. oxysporum f.sp. radicis cucumerinum- Forc, και Botrytiscinerea. Μέρος των μηχανισμών που εμπλέκονται στην επαγωγή της ανάπτυξης και της άμυνας των φυτών μελετήθηκαν με ανάλυση της έκφρασης γονιδίων σε φυτά αγγουριάς και τομάτας που δέχθηκαν εφαρμογή με τον BsMBI600. Οι μετρήσεις της γονιδιακής έκφρασης έδειξαν την ενεργοποίηση γονιδίων που σχετίζονται με την άμυνα του φυτού έναντι φυτοπαθογόνων μικροοργανισμών (PR-proteins, POD, GluA). Επίσης καταγράφηκε υπερέκφραση γονιδίων που σχετίζονται με παραγωγή ουσιών όπως αιθυλένιο, ιασμονικο οξύ και κινάσες, που είναι γνωστό ότι λειτουργούν ως σήματα της επαγωγής διασυστηματικής αντοχής. Τέλος, καταγράφηκε υπερέκφραση γονιδίων που σχετίζονται με την παραγωγή φυτοορμονών, οδηγώντας στο συμπέρασμα πως η εφαρμογή με τον βιολογικό παράγοντα συμβάλει στην επαγωγή της ανάπτυξης των φυτών τομάτας και αγγουριάς μέσω της ενεργοποίησης παραγωγής ενδογενών φυτοορμονών. Τέλος μελετήθηκε η αλληλεπίδραση μεταξύ του βιολογικού παράγοντα και του φυτοπαθογόνου μύκητα B. cinerea. Οι αντιμικροβιακές ουσίες που παράγονται από τον BsMB600 συμβάλλουν στον άμεσο τρόπο δράσης του έναντι του παθογόνου. Μέσω μεταγραφωμικής ανάλυσης σε στέλεχος του B. cinerea μετά την έκθεση του στις παραπάνω ουσίες, ταυτοποιήθηκαν μηχανισμοί που σχετίζονται με την αποτοξικοποίηση ξενοβιοτικών ουσιών (ABC, MFStransporters). H διαγραφή του γονιδίου mfs1 σε στελέχη τουμύκητα, έδειξε πως επηρεάζονται ορισμένες λειτουργίες του, αλλά και η αντιμετώπιση του από τον βιολογικό παράγοντα.

ASSESSMENT OF THE INTEGRATED CHITINASE GENE STABILITY IN WHEAT LINES AFTER BIOBALLISTIC TRANSFORMATION

The synthesis of PR-proteins (pathogenesis related proteins), the most studied of which are chitinases and β-1,3-glucanases, occurs in response to infection with pathogens in plants. Information about the exact role of individual PR proteins within plant immunity makes it possible to use certain specific antifungal proteins for the development of transgenic plants with increased resistance to fungal diseases. At the same time, it is important not only to obtain a plant with the desired trait, but also to fix in it the stable expression of the transferred gene and the inheritance of the acquired trait in generations. Herein we have studied the stability of the chitinase gene insertion in T1, T2 and T3 generations of transformed wheat lines obtained by the method of cis-gene transfer. Primary transformed regenerant plants were obtained as a result of bioballistic transformation of the chitinase gene into immature wheat germ of Saratovskaya 29 and Kazakhstanskaya 19 varieties. Following screening of regenerant plants by PCR for the presence of the target gene made it possible to select 6 lines presumably carrying the insert based on variety Saratovskaya 29 and 2 lines based on variety Kazakhstanskaya 19. The seed material of the selected regenerant plants was cultivated in soil conditions and the seeds of the T1 generation were obtained. DNA amplification of 8 selected lines St-29№25, St -29№43, St-29№44, St-29№33, St-29№26, St-29№35, Кz-19№1, Кz-19№2 with specific primers revealed insert-carrying lines, partially cleaved lines and lines with a high degree of insert instability. According to the results of the T2 and T3 generations PCR analysis, a complete absence of insertion in the St-29№35 and St-29№33 lines was revealed, a partial cleavage of the trait in the St-29№43 and Kz-19№2 lines was revealed, and the stable inheritance of the chitinase gene in four lines St-29№25, St-29№44, St-29№26 and Kz-19№1.was confirmed. These lines were selected as promising for in-depth study of their resistance to fungal diseases and further replication.

ACACIA AURICULIFORMIS-EXTRACT SYNTHESIS PR-PROTEINS DEVELOPED POTENTIAL BIOMEDICINES-VACCINE AGAINST OKRA-DISEASES AND COVID-19: IMPROVED SCIENCE TECHNOLOGY COMMUNICATIONS BIO-ECONOMY APPLICATIONS

The pandemic COVID19, effects on global health, educations, clinical research, human civilization, and the economy. So, it has been an urgency to develop proper vaccines against coronavirus. India emphasis on the most nutritious economically-important number-one-consumption-vegetable, okra, used in many human diseases, is naturally infected by different pathogens and significantly reduces production. Though pesticides are the most effective means of control, but they are expensive and toxic to the environment. So, it will require new and more efficient solutions, technologies, products, and methods. Present pre- and post-treatment with biomedicines; Acacia auriculiformis-extract, prepared from the funicles of Acacia auriculiformis A. Cunn., dissolved in sterile tap water at 1mg/10ml, were applied by foliar spray once daily for 15 days @ 10 ml/plant, against Root-Knot (RK), Yellow Vein Mosaic Virus (YVMV) and Okra Enation Leaf Curl Virus (OELCV) diseases of okra, (Abelmoschus esculentus L. Moench) cv.Ankur-40. The soluble root-proteins were separated by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were scanned with a recording densitometer electrophoretic scanner, measuring the molecular weight of proteins. Pre- and post-treatment with high-diluted-biomedicines; Acacia auriculiformis extract, were highly effective in reducing different okra-diseases by synthesis of new pathogenesis-related-protein (PR-proteins), inducing their natural defense-response against pathogens-infection and enhanced their growth as well as protein content of fruits and roots. The high-diluted-biomedicines; Acacia auriculiformis extract, not only used as a potential-biomedical-drugs or vaccine against various plant-diseases, by synthesis of new PR-proteins, increasing natural-defense-response, but also, it may help for the preparation of a vaccine against COVID-19, by increasing immunity and enriching science and technology communications applications, cost-effectiveness and biodiversity conservations as well as bio-economy-applications issues, reviving human civilizations in the old form. And in near future okra may itself be a ‘Potential Biomedicine as well as Vaccine’ and world will return in normal form by defeating COVID-19.

Moment of Bentonite Addition, Co-Addition of Tannins, and Bentonite Type Affect the Differential Affinity of Pathogenesis-Related Grape Proteins towards Bentonite during Fermentation

To test the effect of the moment of bentonite addition, co-addition of tannins, and bentonite type on the differential affinity of pathogenesis-related (PR) proteins towards bentonite during grape must fermentation, three separate experiments were set up. PR proteins in the obtained wines were analyzed by reverse phase and size exclusion high-performance liquid chromatography (HPLC). The most significant reduction of bentonite dose and PR protein concentration was achieved by applying bentonite in the last third of fermentation. Particular thaumatin-like proteins (TLP) and proteins with lower molecular mass in general were more affected than others, while TLPs were more affected than chitinases. Exogenous enological tannins interacted with particular PR proteins, mostly TLPs, and lowered the total bentonite dose required. The combined application of tannins and bentonite in fermentation removed more PR proteins than bentonite alone, but did not achieve a synergistic effect in reducing the bentonite dose. Various bentonite types, including two Na-activated bentonites, an activated Na bentonite with specifically adsorbed silica, and an active Na-Ca bentonite, exhibited differential affinity towards different PR proteins. The results obtained could be used in developing wine fining protocols which combine treatments with complementary affinity for adsorption and removal of PR proteins, and in this way achieve greater efficiency of bentonite fining by reducing its total dose, which is of significant interest to the wine industry.

Salicylic acid primed defence response in octoploid strawberry (Benihoppe) leaves induces resistance against Podosphaera aphanis through enhanced accumulation of proanthocyanidins and up-regulation of pathogensis-related genes

Background Podosphaera aphanis , a predominately biotrophic fungal pathogen, causes significant yield losses of strawberry. China is the largest strawberry producer in the world, and selecting for powdery mildew-resistant cultivars is desirable. However, the resistance mechanism against P. aphanis in the octoploid strawberry remains unclear. Results To understand the molecular resistance mechanisms, we inoculated strawberry with P. aphanis , and examined the expression profiles of candidate genes and the biochemical phenotypes in strawberry leaves of two groups. The unigenes obtained from salicylic acid (SA)-untreated (SA–) and treated (SA+) leaves resulted in a total of 48,020 and 45,896 genes, respectively. KEGG enrichment showed that phenylpropanoid biosynthesis, plant–pathogen interaction, and plant hormone signal transduction pathways were enriched to a noticeable extent. Comparative analysis demonstrated that genes associated with the SA signalling pathway were significantly upregulated in the strawberry– P. aphanis interaction. In particular, the genes FaTGA , FaDELLA , and FaJAZ negatively regulating salicylic acid SA-responsive genes, whereas FaNPR1 , FaWRKY33 , FaWRKY70 , and FaMYC2 positively regulated SA-responsive genes, leading to increased expression of SA-responsive genes compared to a significant decline in expression of jasmonic acid-responsive genes. Conclusions This study describes the role of total flavonoid content, proanthocyanidins (PAs), pathogenesis-related (PR) proteins, SA, and transcription factors in regulatory model against P. aphanis , which coincided with an early activation of defence, leading to the accumulation of PAs and the production of PR proteins.