Functional characterization of wheat, fusarium head blight resistance (QTL) «Fhb1» based on non-target metabolomics and proteomics

Fusarium head blight (FHB) caused by Fusarium graminearum is a dreadful disease of wheat (Triticum aestivum L.). Host resistance to FHB in wheat is quantitatively inherited. Though more than 100 QTLs have been identified, only a few have been validated. However, the resistance mechanisms governed by these QTLs are poorly understood. A type II FHB resistance QTL Fhb1 is the most consistent and largest effect QTL in wheat against FHB spread in wheat. Non-targeted metabolic and proteomic profiling of wheat near isogenic lines (NILs) with resistant and susceptible Fhb1 alleles was used to functionally characterize Fhb1 using a high resolution LC-MS. The Fhb1 from a moderately resistant cultivar Nyubai was associated with cell wall thickening, mainly at the rachis, due to deposition of hydroxycinnamic acid amides (HCAAs), phenolic glucosides and flavonoids. A hypothetical protein coding gene (GenBank: CBH32656.1) near Fhb1 locus was putatively identified as hydroxycinnamoyl transferase, which catalyzes the biosynthesis of HCAAs. Deoxynivalenol (DON) accumulation was high in both the NILs, eliminating DON detoxification as a mechanism associated with Fhb1 (Chapter III). For additional confirmation, the Fhb1 resistant allele, from a highly FHB resistant cultivar Sumai-3 was profiled. Even though the DON accumulation was low in resistant NIL, the detoxification of DON by host UDP-glycosyltransferase was moderately high in both the NILs, with no significant difference. Interestingly, unlike in the resistant NIL, constitutively present glycerophospholipids were absent in the susceptible NIL following pathogen inoculation due to degradation of membrane. Membrane degradation was caused due to programmed cell death as evidenced by DNA laddering in the susceptible NIL. A locus TAA_ctg0954b.00390.1 was identified as an Fhb1 candidate gene that contains a calmodulin binding motif and two nucleolar localization signal motifs and hence re-annotated as calmodulin binding protein (TaCaMBP_Fhb1). The TaCaMBP_Fhb1 is induced following pathogen infection, binds to Ca2+ bound calmodulin, and triggers Ca2+ signalling cascade including transcriptional activation of endonucleases that cleaves the genomics DNA and cause programmed cell death. The resistant allele of TaCaMBP_Fhb1 lacks part of the promoter region and is non-functional in triggering Ca2+ signalling. While the susceptible allele of TaCaMBP_Fhb1, with functional promoter region is capable of triggering Ca2+ signalling and programmed cell death. The necrotrophic pathogen F. graminearum feeds on the dead tissue, multiply in the host and produce more DON, following a repeated cycle in the susceptible genotype (Chapter IV). The wheat resistance mechanisms against FHB were further confirmed, based on metabolic profiling of rachis, from a resistant cultivar Sumai-3 and a susceptible cultivar Roblin, for resistance against spread of a trichothecene producing (Wild: FgTri5+) and a trichothecene non- producing (mutant: FgTri5-) isolates of F. graminearum. The wild isolate repressed several host resistance mechanisms in both the cultivars due to production of DON. The FHB resistance to spread in Sumai-3 was mainly because of increased cell wall thickening, especially at rachis, due to deposition of lignin, HCAAs and flavonoids, and partially, due to induced RR metabolites which in turn reduced the fungal biomass and toxin biosynthesis. The resistance was not attributed to DON detoxification by UDP-glycosyltransferase, as it was not significant in both the cultivars confirming our previous studies (Chapter V). The resistant alleles of two Fhb1 candidate genes, identified in this study, can be suitably stacked into genome of elite cultivars to enhance FHB resistance in wheat.La fusariose de l'épi est une maladie fongique attaquant le blé (Triticum aestivum) induite par Fusarium graminearum. La fusariose cause de sévères pertes économiques dues à la réduction de la qualité et des rendements suite à la contamination par les mycotoxines trichothecene. La résistance du blé face à la fusariose est un trait quantitatif. Plus de 100 LCQ on été identifiés et un petit nombre a été validé. Cependant, les mécanismes de résistance gouvernés par ces LCQ sont peu connus. Fhb1 est le LCQ le plus consistent qui produit le plus grand effet face à la fusariose du blé. Une caractérisation fonctionnelle à l'aide d'un LC-MS à haute résolution de lignées isogéniques avec ou sans l'allèle susceptible Fhb1 a générée des profils de métabolites non ciblés ainsi que protéomique. Le Fhb1 d'un cultivar modérément résistant, Nyubai, a été associé avec le développement de la paroi cellulaire plus épaisse, surtout au niveau du rachis due à la déposition d'acides amides hydroxycinnamic (HCAAs), de glucosides phénolique ainsi que de flavonoïdes. Le gène codant pour une protéine hypothétique (GenBank: CBH32656.1) située près du locus Fhb1 a été identifiée comme étant possiblement une hydroxycinnamoyle transférase. Cette protéine déclencherait la biosynthèse de HCAAs. L'accumulation de DON a été plus élevée dans les deux lignes isogéniques. La détoxification de DON est un mécanisme associé avec Fhb1 (Chapitre III). Pour confirmer, l'allèle Fhb1 la résistance du cultivar Sumai-3 a été profilé. Contrairement aux lignes iso géniques, aucune présence constitutive de glycérophospholipides, n'a été détectée chez les lignées susceptibles en raison de la dégradation des membranes. La dégradation de membrane s'est avérée être causée par la mort cellulaire programmée comme le démontre le patron de dégradation de l'ADN de la variété susceptible NIL. Le locus TAA_ctg0954b.00390.1 fut identifié comme candidat pour le gène Fhb1 qui contient un domaine de liaison à la calmoduline et deux signaux de localisation nucléaire. Ce dernier fut donc annoté en tant que protéine de liaison à la calmoduline (TaCaMBP_Fhb1). TaCaMBP_Fhb1 est induit suite à l'infection du pathogène pour ensuite se lier à la calmoduline liée au Ca2+ pour ensuite initier une cascade de signaux qui inclut l'activation transcriptionnelle d'endonucléases qui clivent l'ADN génomique causant ainsi la mort cellulaire programmée. L'allèle résistante de TaCaMBP_Fhb1 présente une délétion au niveau du promoteur ce qui la rend non fonctionnel pour l'activation du signalement Ca2+ impliqué dans la mort cellulaire programmée. Le pathogène nécrotrophe F. graminearum se nourrit des tissus morts, se multiplie et produit plus de DON pour faciliter l'infection; perpétuant ainsi un cercle vicieux chez le génotype susceptible (Chapitre IV). C'est résultats on été confirmés à l'aide d'un profilage métabolique des rachis de la lignée résistante Sumai-3 et la lignée susceptible Roblin lors de l'infection avec (Wild : FgTri5+) trichothécène producteur et (Mutant :FgTri5- ) trichothécène non producteur qui sont deux isolats de F. graminearum. L'isolat producteur est parveu à inhiber plusieurs mécanismes de résistance de l'hôte dans les deux cultivars grâce à la production de DON. La résistance FHB à l'infection dans Sumai-3 était principalement lié à l'augmentation des parois cellulaires particulièrement au niveau des rachis à cause de la déposition de lignine, HCAAs et de flavonoïdes et partiellement due à l'augmentation des métabolite RR qui réduisent la biomasses des champignons ainsi que la synthèse des toxines. La résistance n'a pas été attribuée à détoxification de DON par l'UDP-glycosyltransferase, puisque les résultats étaient similaires dans les deux cultivars (Chapitre V). Les allèles résistants, des deux gènes candidats Fhb1 identifiés dans cette étude, pourraient-être ajoutés au génome de cultivars élites de blé pour augmenter leur résistance au FHB

Nanotechnology for the detection of plant pathogens

abstract: Plant pathogens are the important yield-limiting factors, which significantly reduce crop productivity globally, posing serious problems for food security and continues to be the biggest agricultural concern in the world. Even though chemical treatment is still the primary strategy for reducing the incidence of plant disease, their repeated application can cause the pathogens to become less susceptible. Over spraying can also pollute the environment and significantly affect soil microbiota. Therefore, to ensure agricultural sustainability and food security, efficient diagnostic techniques for the rapid identification of plant pathogens in the early stages of infection become crucial. Many molecular approaches for rapid plant pathogen detection have been developed to achieve this goal. However, they are time-consuming, costly, require skilled operators, and are generally unsuitable for in-situ analysis. Plant protection is feasible when any of the nanotechnology tools like microneedle patches, nanopore sequencing, nano barcoding, nano biosensors, quantum dots, nano diagnostic kit equipment, metal nanoparticles, miRNA based nanodiagnosis, and array based nano sensors is used for plant pathogen diagnosis. As they emerge as a potential tool to improve the sensitivity, accuracy, and rapidness of plant pathogen identification, and facilitate high-throughput analysis. The current review focuses on the use of nanotechnology for more quick, inexpensive, and accurate plant pathogens diagnosis

<i>F. graminearum</i> induced shunt phenylpropanoid pathway showing the synthesis of hydroxycinnamic acid amides following the conjugation of amides synthesized from amino acids with hydroxycinnamic acid CoA thioesters (Compounds in bold/red letters are detected in the study).

<p>Pathway adapted from <a href="http://pmn.plantcyc.org/ARA/NEW-IMAGE?type=PATHWAY&object=PWY-5473" target="_blank">http://pmn.plantcyc.org/ARA/NEW-IMAGE?type=PATHWAY&object=PWY-5473</a>, 5474, 40.</p

Resistance related induced (RRI) proteins identified in wheat NIL with resistant allele derived from Nyubai inoculated with <i>F. graminearum.</i>

@<p>RRI = (RP/RM)/(SP/SM). RP: resistant NIL with pathogen inoculation, RM: resistant NIL with mock inoculation, SP: susceptible NIL with pathogen inoculation, SM: susceptible NIL with mock inoculation.</p

Putatively identified FHB resistance related metabolites, other than phenylpropanoids, in rachis and spikelets of resistant wheat NIL with <i>Fhb1</i> upon <i>F. graminearum</i> or mock inoculation.

£<p>Detailed compound identification is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040695#pone.0040695.s005" target="_blank">Table S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040695#pone.0040695.s006" target="_blank">S2</a>.</p>@<p>Fold change calculation: were based on relative intensity of metabolites, RRC =  RM/SM, PRr =  RP/RM, RRI =  (RP/RM)/(SP/SM); PRr;RRI  =  RP/RM, PRr fold change is reported for the metabolites detected only in NIL-R.</p><p>*<i>t</i> test significance at <i>P</i><0.05, <b>**</b><i>t</i> test significance at <i>P</i><0.01, *** <i>t</i> test significance at <i>P</i><0.001.</p><p>NIL =  Near isogenic line, Da: Daltons, RRC  =  Resistance related constitutive, RRI  =  Resistance related induced, PRr = Pathogenesis related metabolite detected in resistant NIL; RP =  resistant NIL with pathogen inoculation, RM =  resistant NIL with mock inoculation, SP =  susceptible NIL with pathogen inoculation, SM =  susceptible NIL with mock inoculation.</p

Laser scanning confocal micrographs of rachis sections, exhibiting secondary cell wall thickening, due to: a) HCAAs (blue fluorescence) and b) flavonoids (yellow fluorescence).

<p>RP is resistant NIL with <i>F. graminearum</i> (pathogen) inoculation, RM is resistant NIL with mock inoculation, SP is susceptible NIL with <i>F. graminearum</i> inoculation, SM is susceptible NIL with mock inoculation, mx is meta xylem, px is protoxylem, ph is phloem, c is cortical cells.</p

Canonical discriminant analysis of significant (<i>P</i><0.05) metabolites in: (a) rachises and (b) spikelets of wheat NILs with resistant and susceptible alleles of <i>Fhb1</i> upon <i>F. graminearum</i> or mock inoculation.

<p>Where, RP is <i>F. graminearum</i> inoculated NIL-R, RM is mock-inoculated NIL-R, SP is <i>F. graminearum</i> inoculated NIL-S, SM is mock-inoculated NIL-S.</p

Relative transcript expression of <i>Triticum aestivum</i> agmatine coumaroyl transferase (<i>TaACT</i>) at 72 hpi in wheat NILs with resistant and susceptible alleles of <i>Fhb1</i> upon <i>F. graminearum</i> and mock inoculation.

<p>RP is resistant NIL with <i>F. graminearum</i> inoculation, RM is resistant NIL with mock inoculation, SP is susceptible NIL with <i>F. graminearum</i> inoculation, SM is susceptible NIL with mock inoculation.</p

Accumulation of resistance indicator (RI) metabolites in wheat NILs with resistant and susceptible alleles of <i>Fhb1</i> inoculated with <i>F. graminearum</i> (a) Accumulation of DON, 3ADON and D3G; (b) Regression models to predict proportion of total DON converted to D3G (PDC) as a function of total DON produced (TDP).

<p>Where, DON  =  deoxynivalenol, 3ADON  = 3-acetyl-deoxynivalenol, D3G  =  DON-3-<i>O</i>-glucoside, TDP  =  total DON produced, PDC  =  proportion of DON converted to D3G.</p

Fusarium head blight resistance related metabolites identified in rachis and spikelets of wheat NIL with resistant <i>Fhb1</i> allele upon <i>F. graminearum</i> or mock inoculation.

£<p>Detailed compound identification is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040695#pone.0040695.s005" target="_blank">Table S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040695#pone.0040695.s006" target="_blank">S2</a>.</p>@<p>Fold change calculation: were based on relative intensity of metabolites, RRC =  RM/SM, PRr =  RP/RM, RRI =  (RP/RM)/(SP/SM); PRr;RRI  =  RP/RM, PRr fold change is reported for the metabolites detected only in NIL-R (qualitative) as the RRI fold change would be infinity.</p><p>*<i>t</i> test significance at <i>P</i><0.05, <b>**</b><i>t</i> test significance at <i>P</i><0.01, *** <i>t</i> test significance at <i>P</i><0.001.</p><p>NIL is Near isogenic line, Da: Daltons, RRC is Resistance related constitutive, RRI is Resistance related induced, PRr is Pathogenesis related metabolite detected in resistant NIL; RP is resistant NIL with pathogen inoculation, RM is resistant NIL with mock inoculation, SP is susceptible NIL with pathogen inoculation, SM is susceptible NIL with mock inoculation.</p