Identification and molecular characterization of flavonoid biosynthetic genes and their expression analysis in wheatgrass (Triticum aestivum L.) during leaf rust infection

doi:10.21203/rs.3.rs-2975149/v1

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Identification and molecular characterization of flavonoid biosynthetic genes and their expression analysis in wheatgrass (Triticum aestivum L.) during leaf rust infection

https://doi.org/10.21203/rs.3.rs-2975149/v1

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Wheat (Triticum aestivum L.) is a major staple food worldwide. Puccinia triticina. forms infectious urediniospores causing leaf rust disease in bread wheat leading to an annual yield loss of ~15% globally. Evolution of new virulent strains and ability of urediniospores to traverse long distances in air pose a challenge on the prevailing leaf rust control techniques. Therefore, significant knowledge is required about the genes in wheat plants that can restrict disease development.Contemporary studies indicate that flavonoid biosynthetic genes are involved in transcriptional and post-transcriptional gene regulation, growth, responses to environmental stimuli and signal transduction. We were able to identify flavonoids like apigenin and luteolin that were synthesized in wheat plants only after leaf rust infection In silico identification of contigs from four SOLiD-SAGE libraries and their functional annotation depicted the involvement of secondary metabolism pathways in retort to the disease development. The flavonoid biosynthetic pathway was discerned through KEGG mapping of the identified contigs and the key genes like Chalcone synthase, Flavanone 3-dioxygenase and Anthocyanidin synthase were characterized. Expression analysis of these genes at varied time points post pathogen infection on both resistant and susceptible wheat Near-Isogenic Lines, revealed their association with development, metabolism, and defense response regulation. Expression of these genes decreased significantly during pathogenesis in susceptible wheat plants compared to the resistant plants, indicating the transition in expression of flavonol accumulation possibly to combat leaf rust disease progression. Consequently, this study focuses on investigating flavonoid biosynthetic genes in wheat and their response during leaf-rust pathogenesis.

Flavonoid dynamics

flavonoid biosynthetic genes

leaf rust

wheat

LC-MS/MS

qRT-PCR

Wheatgrasses are young, germinated wheat shoots of 9–15 days that are proclaimed to have numerous nutraceutical properties in traditional medicines across the world through modulating oxidative stress. Leaf (brown) rust is one of the most damaging foliar fungal diseases of wheatgrass (Triticum aestivum L.) worldwide caused by Puccinia triticina Eriks. (Roelfs et al., 1992). It initially attacks leaf blades, followed by leaf sheath in highly susceptible cultivars. An early occurrence of leaf rust infections in wheat causes yield losses higher than 50%, sometimes it even results in greater losses than any other rusts (Huerta-Espino et al., 2011). Leaf rust infection can be controlled effectively by fungicides; however, it is uneconomical and not environment friendly (Roelfs et al., 1992). About 80 leaf rust resistance (Lr) genes had been designated in wheat, its related species and progenitors (McIntosh et al., 2000; Kumar et al., 2021) many of which were introgressed and pyramided through resistance crop breeding programmes. But the resistance often breaks as the pathogen evolves rapidly (Dixon and Paiva, 1995).

Flavonoids are a class of polyphenolic, naturally occurring secondary metabolites in higher plants that have been reported to play multiple roles in plant responses against biotic and abiotic stresses (Agati and Tattini, 2010). Flavonoids have potential antioxidant properties too that significantly contribute to the scavenging of free radicals produced in plants via metabolic constraints or due to abiotic and biotic stress (Lu et al., 2017; Adhikary et al., 2021). Different flavonoids have distinct and separate biological functions, like protection against ultraviolet (UV) radiation and phytopathogens, signaling during nodule formation, auxin transport as well as responding to stresses (Nakabayashi et al., 2014). They are biosynthesized via the phenylpropanoid pathway, converting phenylalanine into 4-coumaroyl-CoA, which ultimately flow into the flavonoid biosynthesis pathway (Falcone et al., 2012) involving Chalcone synthase (CHS), Flavanone 3-hydroxylase (F3H), Dihydroflavonol 4-reductase (DFR) and Anthocyanidin synthase (ANS) genes and are administered by MYB transcription factors (Fig. S1) (Shi and Xie, 2014; Tian et al., 2015; Xu et al., 2015). In plants regulation of the accumulation of flavonoid components, such as flavone, flavonol, flavanols, procyanidins and anthocyanins is governed through this network of genes.

One of the important subgroups of flavonoids are anthocyanins which play numerous roles, particularly in pigmentation of most flowers, fruits, leaves and seeds. Anthocyanins show protective properties against UV rays by absorbing the blue-green spectra as well as preventing cell damage from photoinhibition or high stress. Such protective effects were observed in red juvenile leaves, autumn leaves, and broad-leaf evergreen plants that turn reddish during elevated exposure to winter sun (Fogelman et al., 2015; Peixoto et al., 2016). The phenotypic appearance of yellow/brown spots on different plant organs as well as on leaf surfaces is due to the accumulation of flavonoids which potentially depicts environmental stress or exposure to pathogen infection. When confronted with various stresses plants often produce pigments in response as an early symptom which can be utilized for diagnosis and prevention strategies (Mierziak et al., 2014; Yang et al., 2016). However, the role of these different phenotypic pigmentation patterns on different plant parts is sundry and the underlying mechanism at the molecular level is mostly unexplored.

Earlier studies have also reported that epidermal flavonoids are involved in defense mechanism as epidermal anthocyanin production in cotton leaves is a sign of resistance to bacterial blight by Xanthomonas campestris pv. malvacearum (Kangatharalingam et al., 2002). Wound periderm development in the lesion margins in Eucalyptus globulus in retort to Cytonaema sp. infection is associated with the buildup of flavanols (Eyles et al., 2003). Studies in grapevine leaves and in cucumber epidermal tissues showed involvement of epidermal flavonoids in plant defense mechanism against biotrophic fungi downy mildew (Plasmopara viticola) (Agati et al., 2008; Latouche et al., 2013) and powdery mildew (Podosphaera xanthii) (McNally et al., 2003) respectively. Similarly, wheat rust pathogens being also biotrophic, was assumed that flavonoids might be implicated in response to infection. The latent genetic relationship between leaf epidermal flavonoids and stripe (yellow) rust response in wheat were evaluated by mapping quantitative trait locus (QTL) using 9K single nucleotide polymorphism (SNP) array in a doubled-haploid (DH) population segregating for several stripe rust resistance genes (Emebiri et al., 2019). The results revealed that flavonoids being naturally synthesized during the normal course of plant development but increased in accumulation after pathogen infection detected using Dualex sensor (Treutter, 2006). Such outcomes has increased the interest in exploring the natural processes of defense mechanisms in wheat, as this might contribute to design effective strategies leading towards development of durable resistance.

However, regulation and modification of expression of flavonoid biosynthetic genes and their relationship to metabolite accumulation in response to pathogen infection remain an attractive target for research. In this study, data were retrieved from four SAGE (Serial Analysis of Gene Expression) libraries that were prepared from mock and leaf rust pathogen inoculated near isogenic wheat lines (NILs; with or without seedling leaf rust resistance gene Lr28) and were sequenced using the Next Generation Sequencing (NGS) tool SOLiD3 (Chandra et al., 2016; Singh et al., 2017). Our aim was to explore the role of flavonoid-based defense mechanism to understand the phenomenon underlying wheat-leaf rust interaction in both susceptible and resistant wheat NILs.

The study was designed with five-phase stages comprising of several experimental and analytical steps (workflow outlined in Fig. 1). Phase I involved the germination of wheat near-isogenic line (NIL) seeds and leaf rust pathogen inoculation at two-leaf stage. Phase II involved sampling of leaves at different time points: hours post inoculation (hpi). Phase III comprised of preparation of sampled leaf extracts for estimation of total contents and metabolite profiling using UHPLC-MS/MS at different post inoculation time points. Phase IV was oriented to design expression study of the key flavonoid biosynthesis genes. In Phase V, the collected leaf sample were subjected to morphological and developmental investigation of leaf rust pathogenesis at different hpi.

Plant growth, pathogen inoculation and leaf sample collection

Seeds of NILs of the wheat cultivar HD2329 (seedling leaf rust susceptible, infection type + 3) and HD2329 + Lr28 (seedling leaf rust resistant, infection type 0;) were sown in pots containing soil and farmyard manure (7:1). The Lr28 gene was derived from Aegilops speltoides (Tauschii) to chromosome 4DL of wheat and is effective against all pathotypes of the leaf rust pathogen in India (Cherukuri et al 2005). The pots were maintained in a greenhouse with 22°C daytime temperature and 14°C nighttime temperature with 80% relative humidity and a photoperiod of 16 h day light intensity 800 µEm^− 2s^− 1 and 8 h dark. The seedlings were allowed to grow till two-leaflet stage (Zodoks growth scale 12) and were divided into four different groups for both susceptible and resistant NILs that included: HD2329 (S-MI; susceptible mock inoculated; and S-PI; susceptible pathogen inoculated) and HD2329 + Lr28 (R-MI; resistant mock inoculated; and R-PI; resistant pathogen inoculated). The S-PI and R-PI plants were inoculated with urediniospores of Puccinia triticina pathotype 77 − 5 (121R63-1), which was selected as the experimental pathogen. The pathogen inoculum was prepared by addition of urediniospores and talcum powder (ratio 1:1) and applied smoothly with a paint brush on the leaves (Singh et al., 2012). After inoculation, the pots were placed under constant high humidity (> 90%) condition for 48 h in the dark, to facilitate infection. Thereafter, the plants were maintained in a phytotron under controlled temperature (22°C), relative humidity (80%), and light periods (16 h day with 800 µEm^− 2s^− 1 and 8 h dark; Chandra et al., 2016) at National Phytotron Facility, Indian Agricultural Research Institute (IARI), New Delhi. Leaf samples were collected from five different plants of same treatment at different time points, 0 (just before inoculation), 24-, 48-, 72- and 168-hour post inoculation (hpi), flash frozen in liquid nitrogen and stored at -80°C freezers for further studies. These time points were selected based on earlier leaf rust pathogen-wheat interaction studies conducted in our laboratory (Singh et al., 2017; Kumar et al., 2017). Leaf extracts were prepared from the samples collected at the above-mentioned time points from 10 g (fresh weight, FW) as mentioned in Adhikary et al. (2021). Dry weights of pulverized powders were recorded and resuspended in one milliliter of aqueous methanol (80%) and stored at − 20°C until further analysis for estimation of total phenolic content (TPC) and total flavonoid content (TFC). Anthocyanin content was estimated from fresh leaves collected at the above-mentioned time points.

Estimation of total phenolic content

The TPC of leaf extracts collected at different time points (0, 24, 48, 72 and 168 hpi) from both the isolines were estimated following the method described by Sulaiman et al. (2011) with minor modifications. One milliliter of the extract was added to 0.5 mL Folin Ciocalteu reagent, diluted with 1.9 mL of double distilled water, and incubated at RT for 5 min, followed by addition of 1.6 mL of saturated sodium carbonate (5% w/v). The incubation was continued for another 2 h at RT. The absorbances were recorded using a spectrophotometer at 765 nm for each sample and TPC was expressed as mg of gallic acid equivalent (GAE)/g dry weight.

Estimation of total flavonoid content

The TFC of the same leaf extracts were estimated following the method by Ma et al. (2014). The absorbances were recorded at 415 nm and were expressed as mg of quercetin equivalent (QE)/g dry weight.

Estimation of total monomeric anthocyanin content

Fresh leaf samples (0.5 g) were finely pulverized using liquid nitrogen and were extracted with 1.5 mL mixture of methanol/hydrochloric acid/water (25:5:70, v/v/v). The extracts were placed on a rotary shaker at 150 rpm for 4 h at 32°C, centrifuged at 10,000× g for 20 min at 4°C and the supernatant was collected. Anthocyanin content was determined by recording the absorbance at 525 nm and 657 nm as An = (OD525–0.25 × OD657)/FW and were expressed as mg of 100/g fresh weight (Ma et al., 2014).

Ultra-high-performance liquid chromatography LTQ-XL iontrap MS/MS analysis

The frozen leaf samples (500 mg FW) of both the mock and pathogen inoculated NILs as mentioned above were used for metabolite analysis, collected at three time points (0, 72 and 168 hpi) and stored at -80°C. Preparation of samples and UHPLC-MS/MS procedure was performed exactly as mentioned in Adhikary et al. (2021).

Gene ontology annotation

Four Serial Analysis of Gene Expression (SAGE) libraries (Short Read Archive Biosample number SRA061917) of the same wheat NILs either mock inoculated or inoculated with urediniospores of Puccinia triticina [SAGE1 (S-MI; accession number SAMN01820702), SAGE2 (S-PI; accession number SAMN01820703), SAGE3 (R-MI; accession number SAMN01820704) and SAGE4 (R-PI; accession number SAMN01820705)] were earlier prepared in this lab using SOLiD3 System SAGE kit and sequenced (Applied Biosystems, CA, USA; Chandra et al., 2016; Singh et al., 2017). The retrieved sequences were functionally annotated at KEGG (Kyoto Encyclopedia of Genes and Genomes) using KAAS (KEGG Automatic Annotation Server; https://www.genome.jp/kegg/kaas/; Chandra et al., 2016; Yuan et al., 2018). Sequences assigned with KO identifiers or K numbers after initial screening of SAGE libraries, were BLAST searched against completely annotated Oryza sativa genome sequence as reference using BBH (Bidirectional Best Hit) method to identify similarity between the query and reference sequences with the highest alignment score. The matched sequences were further subjected for pathway annotation and map was obtained using KAAS by the BRITE hierarchy file (Moriya et al., 2007; Chandra et al., 2016; Yuan et al., 2018). The sequences were also functionally annotated with Gene Ontology (GO) terms using CLC Genomics Workbench 21.0 (www.qiagenbioinformatics.com). The annotated results were visualized using Web Gene Ontology Annotation Plot (WEGO; http://wego.genomics.org.cn/; Ye et al., 2018).

Gene identification

The flavonoid biosynthetic genes that were selected from the pathway, were represented with Enzyme Commission (EC) numbers and their names were retrieved from the ExPASy-ENZYME server (https://enzyme.expasy.org). The genes were searched at NCBI (http://www.ncbi.nlm.nih.gov), to obtain Triticum aestivum specific nucleotide sequences that were BLAST searched at EnsemblPlants (release 49, 2020) (https://plants.ensembl.org/index.html) against Triticum aestivum genome (http://plants.ensembl.org/Triticum_aestivum/genome/index). The presence of flavonoid specific genes was confirmed by assigning the sequences to Conserved Domain Database (CDD, https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) and the matched sequences were selected for further studies.

Multiple sequence alignment and construction of phylogenetic tree

The phylogenetic relationship of the identified flavonoid genes of wheat were aligned with protein sequences of flavonoid biosynthetic genes of Oryza sativa, Hordeum vulgare and Zea mays obtained from NCBI using Molecular Evolutionary Genetic Analysis (MEGA) ver. 11. A phylogenetic tree was constructed by Neighbor-Joining method and bootstrap analysis was performed with 1000 replicates (Tamura et al., 2011).

In silico characterization of identified flavonoid biosynthetic genes

In silico characterization like residue length, molecular weight, theoretical pI, aliphatic index and grand average of hydropathicity (GRAVY) were determined using ExPASy ProtParam tool (https://web.expasy.org/protparam/). The Nuclear Localization Signals (NLS) were recorded using NLStradamus (http://www.moseslab.csb.utoronto.ca/NLStradamus) software. The presence of conserved motifs in the sequences were studied using the Multiple Em for Motif Elicitation (MEME) suite 5.0.5 (http://meme-suite.org/index.html). Wheat MicroRNAs catalogued at miRBase (http://www.mirbase.org/index.shtml) targeting the identified flavonoid genes were predicted using professional small RNA (psRNA) target analysis server (http://plantgrn.noble.org/psRNATarget/) to determine whether these genes are also post-transcriptionally regulated or not. The 1500–2000 bp upstream region of the genes were considered to contain regulatory regions and were submitted to Plant Cis-Acting Regulatory Elements (PlantCARE, http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) database for detection and analysis of various regulatory elements. The synonymous (Ks) and non-synonymous (Ka) substitution rates were calculated for the identified genes in PAL2NAL server (http://www.bork.embl.de/pal2nal/) and the divergence period was determined using the equation T = Ks/(2 x 9.1 x 10^− 9)x10^− 6 million years ago (Suyama et al., 2006).

The interaction network between the query proteins and predicted functional partner proteins were displayed as a network using STRING database v11.0 (https://string-db.org/). The in-silico expression study of identified genes in different tissues and organs under different development stages and various stress conditions was visualized through heat map using the flexible Wheat Expression browser, expVIP (http://www.wheat-expression.com/). For synteny analysis BLASTN search was performed of the query sequences using EnsemblPlants (https://plants.ensembl.org/index.html) considering the criteria of alignment score being the highest of the rest of the homologues; and lowest E-value. Each gene was mapped using PhenoGram software (https://ritchielab.org) with other monocot species such as rice and on the hexaploid wheat chromosome. The BLAST scores of the homology sequences of respective gene sequences on Triticum aestivum were visualized using Circos-0.68 (http://circos.ca/).

Gene expression analysis using quantitative Real-Time PCR

Total RNA was isolated using TRI Reagent (Sigma-Aldrich) from the mock and pathogen inoculated leaves of both wheat NILs sampled at varied infection time points (Dutta et al., 2019). Complementary DNA was prepared from 1µg of isolated RNA using RevertAid First Strand cDNA synthesis kit (Thermofisher) as suggested by the manufacturer. Gene specific primers were designed for the identified flavonoid biosynthetic genes using PrimerQuest (https://eu.idtdna.com/PrimerQuest). The relative expression levels of these genes in both isolines were evaluated by qRT-PCR performed using a Rotor-Gene Q Real Time PCR Cycler (Qiagen, USA). The reactions were carried out in final volume of 20 µl, containing 100 ng of cDNA, 0.2 pmol/µl of gene specific forward and reverse primers and 10 µl PowerUp SYBR Green Master Mix (Applied Biosystems). Each reaction was performed with three replicates including non-template as control. The assay conditions included initial denaturation at 95°C for 5 minutes, followed by 50 cycles of 95°C for 10 seconds, 60°C for 45 seconds and 72°C for 90 seconds; and melt-analysis at 65°C to 95°C for 90 seconds. Gene expression levels were computed relative to the expression of the reference gene wheat GAPDH (Accession No. AF521191) at same time points using the 2^−∆∆CT method (Paolacci et al., 2009).

Specimen preparation and Scanning Electron Microscopy (SEM)

Leaves were collected at 0, 72 and 168 hpi from mock and pathogen inoculated wheat NILs, cut into 3×3 mm squares and fixed in 3% glutaraldehyde in 0.05 M sodium cacodylate buffer (pH 7.2) for 24 h. The leaf segments were then washed twice in the same buffer, postfixed for 2 h in 2% osmium tetroxide buffer and dehydrated in a graded ethanol series (Hu and Rijkenberg, 1998). Specimen samples for morphological imaging were mounted on stubs coated with thin film of platinum that was placed inside the microscopic chamber of a scanning electron microscope, (Jeol, JSM-6390LV; Japan), operating at 10 kV. Analysis of infection and presence of different infection structures at various time points were studied in both susceptible and resistant wheat lines from the obtained images.

Statistical analysis

Statistical analysis was performed for each variable for the different treatment groups. Data were expressed as mean ± standard deviation (n = 3) for TPC, TFC and anthocyanin content using Graph Pad PRISM version 5.01 (San Diego, CA, USA). For gene expression study, data of qRT-PCR were analyzed using repeated measurements in -ΔCt model and were expressed as mean ± standard deviation (n = 3) using OriginPro 9.0 (Northampton, Massachusetts, USA). Furthermore, one-way and two-way ANOVA followed by Dunnett's and Bonferroni’s multiple comparison tests was conducted for group differences. LC-MS/MS flavonoid-related metabolic profiles were obtained and described by multivariate statistics analysis through the typical steps of normalization using Principal Component Analysis (PCA) and heat map approaches after recovering the peak intensity of each m/z feature of the positive mode ionization-derived TICs. The resulting matrix consistesd of the identified compounds at different time points versus variables (i.e., features labelled as m/z) was exported to .csv format and statistically analyzed through MetaboAnalyst 5.0 (McGill University, Quebec, Canada).

Determination of Total Phenolic Content (TPC), Total Flavonoid Content (TFC) and anthocyanin content

TPC, TFC and anthocyanin content significantly (p < 0.001) increased in leaves of both the wheat isolines with respect to disease progression after pathogen inoculation (Fig. 2a-c). Though both the isolines showed augmented TPC and TFC accumulation under disease progression, but major increase (p < 0.01) was in S-PI at 24 hpi compared to 0 hpi. However, continuous increase (p < 0.01) in TPC and TFC build up was observed all through the course of the study in both NILs. Gradual augmentation in anthocyanin content was also observed in both S-PI and R-PI, though the escalation was much sharper (p < 0.001) during 48 hpi to 72 hpi.

Flavonoid profiling by LC-MS/MS in leaves of susceptible and resistant wheat NILs

LC-MS/MS precursor ion scan method was applied to determine the dynamics of phenolic and flavonoid compounds present in the aqueous methanolic leaf extracts of susceptible and resistant wheat NILs under mock (0 hpi) and post leaf rust infection condition (72 hpi and 168 hpi). The main focus was to identify flavonoid conjugates using the pseudo MS³ method that provides structural elucidation about the aglycones and aid in analyzing their fragmentation patterns for characterizing the flavonoid classes specifically generated due to leaf rust. Flavonoids like apigenin and luteolin were mainly identified in the extracts of both wheat NILs only after pathogen inoculation. The total ion chromatogram of the extracts was obtained by full scan mode under positive electrospray ionization (ESI) condition. The identified compounds were clustered based on their presence at 0, 72 and 168 hpi through principal component analysis (PCA; Fig. 3a). The LC-MS/MS data were visualized through a heat map displaying the accumulation of compounds that varied with respect to different time points (Fig. 3b). The mass spectrometric data of the identified compounds along with their classification in both NILs at selected time points as well as briefs the experimental m/z, molecular weight, molecular formula and retention time are provided in Supplementary Table S1. Flavonoids normally accumulate in plants as O-glycosylated derivatives, whereas flavonoids belonging to Poaceae family are characterized predominantly as flavone-C-glycosides (Wojakowska et al., 2013; Piasecka et al., 2015).

Gene annotation

The contigs from the SAGE libraries were subjected to KAAS analysis that assigned the sequences with K numbers after the initial screening using GHOSTX (http://www.bi.cs.titech.ac.jp/ghostx/). The results displayed 200 sequences from SAGE 1, 124 sequences from SAGE 2, 105 sequences from SAGE 3 and 34 sequences from SAGE 4 (Supplementary Table S2). These filtered contigs, assigned with K numbers, were subjected to gene ontological annotation. Further, WEGO annotation was carried out with these sequences to depict their molecular functions, cellular component, and biological processes along with the annotation scores (Supplementary Fig. S1 a-d). Under biological process, the annotated contigs divulged to be involved in immune system process signaling, biological adhesion, carbon utilization, cell proliferation and different organ developmental processes. Under the molecular function, contigs were involved in antioxidant activity, transcription regulator activity, catalytic activity and regulation of biological process. Under cellular component, most contigs were cell part, cell, organelle, nucleoid, supramolecular complex, membrane part, membrane, protein-containing complex, organelle parts.

The K numbers assigned to the contigs of SAGE libraries were further subjected to KEGG database and the secondary metabolite pathway was recorded from the list of KEGG pathways, particularly, flavonoid biosynthetic pathway (Fig. 4). Among those, 25 sequences of SAGE 1, 14 of SAGE 2, 13 of SAGE 3 and five of SAGE 4 respectively were involved in the biosynthesis of secondary metabolites. Flavonoid biosynthetic pathway revealed involvement of several enzymes out of which three putative enzymes with EC numbers were selected for further studies. These EC numbers from the flavonoid biosynthesis pathway were assigned in the ExPASy ENZYME server to derive the names of the genes, reaction catalyzed and their descriptions (Supplementary Table S3). A total of 18 sequences corresponding to the flavonoid biosynthesis genes were identified that belonged to Chalcone synthase (2.3.1.74), Flavanone 3-dioxygenase (1.14.11.9) and Anthocyanidin synthase (1.14.20.4). Chalcone synthase (CHS) is the entry point of the flavonoid pathway, which catalyze 4-Coumaroy-CoA and Malonyl-CoA to chalcone, leading the phenylpropanoid pathway towards flavonoids biosynthesis (Ma et al., 2014). Besides CHS, enzymes such as Chalcone isomerase (CHI), Flavanone 3- hydroxylase (F3H), Anthocyanidin synthase (ANS), Dihydroflavonol 4-reductase (DFR), Flavonol synthase (FLS) and Flavone synthase (FNS) were also determined that are involved in the pathway leading to biosynthesis of major flavonoid classes which aided for the selection of enzymes (Lu et al., 2017) (Supplementary Fig. S2). Therefore, these three genes CHS (initial gene of the pathway), F3H (gene in the middle of the pathway) and ANS (at late stage of the pathway) were selected to characterize and discern their roles during leaf rust pathogenesis.

In silico characterization of the identified flavonoid biosynthesis genes

In silico characterization studies of the 18 identified sequences were carried-out for unraveling their functional attributes. The physico-chemical parameters of the identified proteins demonstrated considerable variations in length, molecular weight, pI and grand average of hydropathicity (GRAVY) values (Supplementary Table S4). All 18 identified amino acid sequences were predicted to have nuclear localization signal (NLS) sequence (Supplementary Table S5). The prediction of NLS depicts stretches of amino acid tags which helps in trafficking of the proteins from cytoplasm into nucleus.

The psRNA target tool predicted 26 flavonoid biosynthetic gene sequences that were targeted by various wheat (tae)-miRNAs (Supplementary Table S6). TaCHS1 and TaCHS2 was targeted by four different miRNAs: tae-miR1121, tae-miR1135, tae-miR9659-3p and tae-miR9776. TaCHS5 was targeted by two miRNAs: tae-miR1130a and tae-miR1130b-3p. TaF3H1 was targeted by tae-miR9781, TaF3H6 was targeted by tae-miR5086 whereas TaF3H3 was targeted by four different miRNAs: tae-miR9670-3p, tae-miR9677a, tae-miR9775 and tae-miR9779. TaANS3 was targeted by tae-miR9670-3p. TaANS1 and TaANS5 was targeted by two different miRNAs i.e., tae-miR531, tae-miR9773 and tae-miR9773, tae-miR1128 respectively whereas TaANS2 was targeted by four different miRNAs: tae-miR1121, tae-miR1847-5p, tae-miR397-5p and tae-miR9781. Though multiple miRNAs targeted the same gene, the target sites on the ORF were different.

Analysis of the regulatory sequences present in the 2000 bp upstream of each gene illustrated various cis-regulatory elements that included light responsive, developmental related, promoter related, flavonoid biosynthesis related, hormone responsive, defense related and environmental stress related elements (Supplementary Table S7). Besides, elements for basal transcription like TATA-box and CAAT-box were detected. The key physiological processes related to the regulatory elements were photoperiod, hormonal response, developmental regulation, and environmental response. Phytohormone responsive abiotic stress elements included P-box, ABRE, TGA, TCA, CGTCA-motif, TGA-element, TGACG-motif and GARE-motif. Among these, CGTCA, P-box and TCA motifs were abundant with average copy numbers of 2.66, 0.22 and 0.27 respectively. These elements are mainly related to methyl jasmonic acid (MEJA), gibberellin and salicylic acid stress. The cis-regulatory elements responsive to other environmental stresses like anaerobic induction, drought inducibility and low-temperature responsiveness were ARE, MBS and LTR with average copy numbers of 1.00, 0.77 and 0.5, respectively. The cis-regulatory element responsive to flavonoid biosynthesis detected was MBSI with an average copy number of 0.77. Defense related elements included TC-rich repeats and WUN-motif with an average copy number of 1.22 and 0.55 respectively. The developmental related elements included CAT-box, O2-site, AT-rich element, GC-motif, GCN4_motif, RY-element and circadian element that are involved in zein metabolism regulation, meristem expression, endosperm expression, and cell cycle regulation respectively. The elements such as CAT-box and GCNA motif was copious. The highest number of cis-acting elements observed were under the developmental related elements suggesting that these identified wheat genes might play critical roles in morphogenesis, differentiation and development.

In order to understand the evolutionary constraints of the selected flavonoid biosynthesis genes of wheat, Ka (non-synonymous substitutions), Ks (synonymous substitutions) and Ka/Ks ratio was determined. Ka/Ks values = 1 denote the neutral drifting of the genes, Ka/Ks > 1 denote the accelerated evolution with positive selection, and Ka/Ks < 1 denote the functional constraint with a negative or purifying selection of genes. The Ka/Ks ratio of the 18 orthologous genes was less than 1, suggesting these genes are undergoing strong purifying selection and they are slowly evolving at protein level in wheat (Fig. 5 and Supplementary Table S8).

The deduced protein sequences comprised of brief patterns of motifs that were originally restored by nature. These motifs refer to an enzyme's active site and structural units that are required for proper protein folding. Hence, the motif sequences encompass basic functional components of molecular evolution (Bailey et. al, 2009). Motifs of CHS, F3H and ANS proteins were identified using MEME which revealed three conserved motifs (Fig. 6). Motif − 1: EGEEPILEEPITFAEMYRRKMERDLDLAKRKKQAKDQLMQQQLQLQQQQQ denotes Leucine Rich Repeats (LRR); Motif-2: ATVLAIGTATPANCVYQADYPDYYFK ITKSDHMADLKEKFKRMCKFNEFH denotes polyketide synthase (type III PKS17) and Motif-3: NYCALCMQFMSNGRFKNADHQAVVNGESSRLSIATFQNPAPDARVWPLAV denotes Naringin (NRG) motifs. The Non-Expresser of Pathogenesis Related (NPR) motifs, found in all sequences, bind with bZIP transcription factors, forming a complex. This complex-formation activates some genes that include Acyl-CoA oxidase (ACO) and Acyl-CoA dehydrogenase that contributes to the synthesis of flavonoids (Garcia-Ponce and Rocha-Sosa, 2000).

Based on the query sequences of Triticum aestivum the evolutionary relationship was analyzed. Neighbor-Joining trees were constructed with the query protein sequences of CHS, F3H and ANS, that were aligned along with Oryza sativa, Zea mays and Hordeum vulgare. The bootstrap values on the branch points of the phylogenetic trees provide the assessments of “confidence” for each clade in the tree and based on homology, the sequences were thereby clustered into three main classes namely Class I, Class II and Class III represented in three different colors (Fig. 7A-C). Figure 7(a) depicts CHS proteins that are clustered in following groups: Class I included eight proteins: TaCHS1, TaCHS2, TaCHS3, TaCHS4, TaCHS5, TaCHS6, HvCHS1 and HvCHS2; Class II included three proteins: ZmCHS3, ZmCHS4 and ZmCHS5 while Class III included eight proteins: ZmCHS1, ZmCHS2, OsCHS1, OsCHS2, OsCHS3, OsCHS4, OsCHS5 and OsCHS6. Based on the phylogenetic tree, five sister gene pairs were formed which included TaCHS3-TaCHS2 showing paralogous relationship and TaCHS6-HvCHS2 showing orthologous sister pair within Class I; ZmCHS3-ZmCHS4 of Class II showed paralogous relationship; OsCHS4-OsCHS5 and ZmCHS1-ZmCHS2 of Class III showed paralogous relationship. Figure 7(b) depicts F3H proteins that are clustered in following groups: Class I included six proteins: TaF3H3, TaF3H4, TaF3H5, TaF3H6, TaF3H7 and HvF3H1; Class II included three proteins: TaF3H1, TaF3H2 and OzF3H2 while Class III included six proteins: OzF3H1, OzF3H3, OzF3H4, OzF3H5, OzF3H6 and ZmF3H1. Based on this phylogenetic tree, four sister gene pairs were formed which included TaF3H3-TaF3H4 and TaF3H5-TaF3H6 of Class I showing paralogous relationships: OzF3H2-TaF3H2 of Class II showing orthologous relationship and OzF3H5-OzF3H6 of Class III showing paralogous relationship. Figure 7(c) depicts ANS proteins that are clustered in following groups: Class I included one sister gene pair: OzANS3-OzANS5 which also showed paralogous relationship; Class II included four proteins: TaANS1, TaANS3, TaANS4, and TaANS5, where TaANS1-TaANS5 showed paralogous sister pairs while Class III included four proteins: OzANS1, OzANS2, OzANS4 and TaANS2.

Further, to better divulge the functional role of flavonoid biosynthesis genes, protein-protein interaction was performed using STRING v. 11 database using plant specific parameters. The search tool predicted other Triticum aestivum proteins which exhibited direct and indirect interaction with the query proteins in the STRING functional association databases (Fig. 8). The interaction revealed 16 nodes with 60 edges. A clustering co-efficient of 0.783 indicated significant interactions among the query proteins. The network analysis predicted five functional partners such as Chi-B1 and Traes_5BL_4F4E0B862.1 which are chalcone-flavonone isomerase that belongs to the chalcone isomerase family whereas Traes_2AS_2E21BFFFB.1 is a putative uncharacterized protein that belongs to chalcone/stilbene synthases family. The other two functional partners i.e., Traes_3AL_197871859.2 and Traes_3B_96D744B6B.1 are putative uncharacterized proteins. It also revealed the pragmatic co-expression protein interactions between Traes_2DS_1BA7B8A2C.1, CHS, Traes_2BL_A3CF87FBF.1, Traes_5BL_4F4E0B862.1, Chi-B1, Traes_2AS_2E21BFFFB.1, Traes_2DL_BC7F606B9.2 and Traes_2DS_B9D014DE1.1 with C- and N-terminal domains that belonged to chalcone synthase, stilbene synthases as well as Chalcone-flavanone isomerase containing 3-Oxoacyl-[acyl-carrier-protein (ACP)] synthase III and FAE1/Type III polyketide synthase-like protein domains. The functional partners identified in the interaction network revealed the CHS proteins to have their functional role in flavonoid biosynthesis mediated signaling pathway.

The in-silico expression study of these genes exhibited high expression in leaves, followed by roots, spikes and grains under normal conditions of growth and development (Fig. 9). In leaves, the expressions of these genes were low during abiotic stress condition whereas high during diseased condition The TaCHS2, TaCHS3, TaF3H2, TaF3H3, TaF3H4 and TaF3H5 genes highly expressed in leaves under high levels of stress-disease while they moderately expressed in roots and spikes. Whereas other genes exhibited diverse expression patterns in tissues and organs during different developmental stages and under biotic stresses. Thus, the uniform and comparatively high expression pattern of TaCHS2 and TaCHS3 genes in leaves demonstrated preclusion under diseased or other biotic stress condition.

Synteny analysis for the comparative genomics study of the genetic loci of query sequences of the three flavonoid biosynthesis genes of wheat on chromosomes of other monocot plants such as Oryza sativa and Hordeum vulgare was studied (Fig. 10). The synteny analysis reveals several clues for the presence of the functional genes. The mapping of identified genes shared more than 80% homology with Hordeum vulgare and Oryza sativa indicating presence of similar NRG and LRR domains inferring the high syntenic relation among these plants. It can be presumed that these genes might have similar functions among all three selected monocot plants.

Expression profiling of flavonoid biosynthesis genes in wheat NILs in response to leaf rust

QRT-PCR was performed to determine the expression of three selected flavonoid biosynthesis genes CHS, F3H and ANS using gene specific primers (Supplementary Table S9) and cDNA prepared from leaves of susceptible and resistant plants collected at varied time points during leaf rust infection progression. As in Fig. 11(a) expression of CHS being less upregulated in S-PI in comparison to R-PI indicating elevated transcription of this gene even prior to infection. As the infection progressed, the expression of the gene at 24 hpi was upregulated in both susceptible and resistant plant types that gradually increased with significant difference (p < 0.05) and was maximum at 168 hpi. No significant difference (p > 0.05) was observed among 24 hpi, 48 hpi, 72 hpi in susceptible plants. On the contrary in R-PI, there were marginal increments with significant difference (p < 0.05) in expression of CHS at 24 hpi, 48 hpi, 72 hpi and 168 hpi respectively.

The F3H gene, depicted a continuous increase in its expression in S-PI throughout the course of pathogen infection; much significant (p < 0.05) upregulation was detected at 48 and 72 hpi and the expression was maximum at 168 hpi. Whereas in R-PI, the gene showed comparatively lower expression and reached optimum expression level at 48 hpi followed by progressively declined expression at 72 and 168 hpi with significant difference (p < 0.05) (Fig. 11b).

The ANS gene expression was also studied at different time-points of leaf-rust progression that exhibited its highest expression in S-PI at 24 hpi and showed comparative down regulation at 48 hpi, 72 hpi and 168 hpi. ANS seems to have significant role at late stages of pathogenesis in R-PI, as its expression was retarded till 48 hpi but demonstrated remarkable increase (p < 0.05) in the expression levels as detected at 72 hpi that even increased at 168 hpi (Fig. 11c).

Scanning of uredinial infection development events at different time points using scanning electron microscope (SEM)

In this study, leaf samples of both susceptible and resistant wheat NILs collected at three different time points of infection (0 hpi, 72 hpi and 168 hpi) were visualized in SEM to study the morphological changes due to uredinial infection by Puccinia triticina. Normal wheat leaf morphology, including trichome and stomata was observed in mock (uninfected) leaves i.e. at 0 hpi of both the wheat NILs (Fig. 12a and 12d). In susceptible plants at 72 hpi, urediniospores were observed along with the formation of appressorium on the stoma and substomatal vesicle (SSV) formation within the stomatal cavity (Fig. 12b). In Fig. 12c, a slender neck formation was also observed at 168 hpi that forms the haustorial mother cell (HMC), firmly attached to the cell wall which subsequently invaginates the host cell plasma membrane to form a balloon-shaped feeding structure, known as the haustorium. Deformation of trichome was also visible in susceptible plants. Whereas in the resistant isoline no infection structures were visible post inoculation; at 168 hpi enlarged trichome was visible (Fig. 12e and 12f).

Flavonoids are small molecules widely distributed in the plant kingdom, which play vital roles in various mechanisms from physiological development and to plant responses to abiotic and biotic stress (Agati et al., 2008). Leaf rust is one of the most widespread biotic stresses in wheat. Wheat plants have evolved a variety of defense mechanisms including production of secondary metabolites such as alkaloids, terpenoids, flavonoids and glucosinolates to combat this stress (Fofana et al., 2005). Flavonoids are constitutively synthesized during the normal growth and development of plant tissues but are also synthesized at elevated levels in response to pathogen infection, wounding and several types of stresses (Treutter, 2006). Research on defense-related epidermal flavonoids determined using a leaf-clip optical sensor in response to biotrophic stripe rust fungi in wheat leaves showed augmented accumulation of epidermal flavonoids in response to the pathogen (Emebiri et al. 2019). The present study also depicted that TPC, TFC and anthocyanin were accumulated more in susceptible wheat plants with respect to progressive exposure to Puccinia triticina. Whereas the resistant wheat plants did not show much differences in TPC, TFC and anthocyanin content during post-infection. In this study a non-targeted LC-MS/MS-based method was applied to discern contrasting flavonoid profiles in the wheat NILs in retort to leaf rust pathogenesis. This broad approach enabled us to access more compounds directly in leaf extracts and efficiently identify a wide spectrum of compounds. The LC-MS/MS analyses demonstrated a shift in metabolite accumulation in susceptible wheat plants. Similar result was also reported in Eucalyptus when inoculated with Austropuccinia psidii (Sekiya et al., 2020). However, flavonoid profiles from susceptible and resistant wheat leaf tissues may also vary due to minor differences in genetic background and varied developmental stages.

Despite the diversity in flavonoids on basis of their functions and structures, all flavonoids are derived through the general phenylpropanoid pathway, one of the most studied pathways in plant secondary metabolism. The Non-Expresser of Pathogenesis Related (NPR) monomers bind with bZIP transcription factors, forming a complex which activates genes like Acyl-CoA oxidase and Acyl-CoA dehydrogenase that contributes in the synthesis of flavonoids (Garcia-Ponce and Rocha-Sosa, 2000). CHS is the entry point of the flavonoid biosynthesis pathway, which catalyze 4-Coumaroy-CoA and Malonyl-CoA to chalcone, leading the phenylpropanoid pathway towards the onset of flavonoid biosynthesis (Ma et al., 2014). Enzymes such as chalcone isomerase (CHI), flavanone 3- hydroxylase (F3H), anthocyanidin synthase (ANS), dihydroflavonol 4-reductase (DFR), flavonol synthase (FLS) and flavone synthase (FNS) are involved in the pathway leading to biosynthesis of major flavonoid classes (Lu et al., 2017). CHS, Chalcone flavanone isomerase (CFI), Chorismate synthase (CS), Iso-chorismate synthase (ICS) genes are involved in various circadian rhythms which gets activated by MYB and MYB-related TFs (Singh et al., 2017). Previous study also acknowledges that flavonoid biosynthetic pathway responds to biotic and abiotic stress in maize (Chiristie et al., 1994), rice (Ithal and Reddy, 2004), Ligustrum vulgare (Guidi et al., 2008), Scutellaria baicalensis (Yuan et al., 2012), and the desert plant Reaumuria soongorica (Liu et al., 2013). Therefore, we investigated the expression profiles of these key enzyme coding genes CHS, F3H and ANS involved in flavonoid biosynthetic pathway in both leaf rust susceptible and resistant wheat near-isogenic plants at different time points post infection.

In the current study, expression of CHS transcript was higher in S-PI compared to R-PI with respect to disease progression. Our results are in agreement as reported by Vasquez-Robinet et al. (2008) on CHS gene to have maximum expression during abiotic and biotic stress conditions. Similar results were also reported in Arabidopsis (Burbulis and Winkel Shirley, 1999), potato (Andre et al., 2009) and Scutellaria baicalensis (Yuan et al., 2012) showing up-regulation of CHS gene with key protective roles against stress. Gradual enhanced expression of F3H transcripts was clearly observed in susceptible wheat isolines after leaf rust infection. Castellarian et al. (2007) in grape berries and Liu et al. (2013) in Reaumuria soongorica reported similar result for F3H gene. Whereas in resistant plants carrying Lr28 gene implicates the potential resistance against pathogenesis thereby inhibiting leaf rust development. ANS genes are involved in late stages of the flavonoid biosynthetic pathway (Ma et al., 2014). The expression pattern of ANS in susceptible plants during initial stages of infection depicts the spotting of color in the pathogen infected tissues due to the accumulation of anthocyanins (Mierziak et al., 2014; Peixoto et al., 2016; Lu et al., 2017). Gradual down regulation of expression of this gene in the present study might indicate the transition in the expression to flavonol accumulation possibly to cope with the expansion of rust disease. These can be corroborated with spot expansion in susceptible plants indicating the activation of defense mechanisms due to pathogen stress. The expression of ANS was up regulated at 24 hpi onwards indicating the potential resistance by Lr28 gene in resistant plants, thereby possible activation of genes of the flavonoid biosynthetic pathway, as was also found in grape berries by Castellarin et al. (2007). Likewise, expression profiles of different genes involved in flavonoid biosynthesis were analyzed by Ma et al. (2014) on two different cultivars of common wheat (Aikang 58 and Chinese Spring) exhibiting up-regulation of TaCHS, TaF3H and TaANS transcripts under abiotic stress. Therefore, we hypothesized that flavonoid accumulation-based defense mechanism operated differently between the susceptible and resistant wheat NILs.

The pathway is regulated by MYB TFs that leads to accumulation of anthocyanins and flavonol. The MYB transcription network results in the variations in essential coloring diversity in the plant kingdom (Tian et al., 2015; Xu et al., 2015). Nevertheless, flavonoids do play protective roles against biotic and abiotic stress but the fine regulation of flavonoid biosynthesis pathway is achieved by combinatorial action of several TFs such as MYB11, MYB12, MYB111 and WD40 involved in the transcriptional regulation of flavonoid biosynthesis genes and plant hormones like abscisic acid (ABA), ethylene (ETH), jasmonic acid (JA) and salicylic acid (SA) which have multiple effects in secondary metabolism (Stracke et al., 2010; Khlestkina et al., 2015). All these underlying factors might have a vital role in imparting resistance mechanism against leaf rust in the resistant wheat isoline (Schijlen et al. 2004).

In order to better understand the temporal development of leaf rust in both the wheat isolines, inoculated leaves collected at different time-points were examined using SEM. Significant differences in leaf morphology of susceptible and resistant wheat plants after infection by the leaf rust pathogen were observed. The appearance of yellow spots were clear evidence of rust disease that were visible on leaves of susceptible plants with blurred edges at 72 hpi because of obvious flavone and flavonol accumulation in the infected regions. At 168 hpi these yellow spots turned brownish in susceptible plants whereas in resistant plants no spots but only flakes were observed. Sekiya et al., (2020) reported germination of spores and production of appressoria by 48 hpi in Eucalyptus grandis. Similarly, in the present study, mature appressoria were typically observed that penetrated inside the leaf tissue of susceptible plants. At 168 hpi, SSVs progressively developed and elongated unilaterally, while remaining firmly attached to the inner epidermal cell surface along with formation of HMCs. On the other hand, response in resistant wheat plants was markedly different from susceptible plants showing no fungal growth indicating presence of strong defense system and pathogenesis could not initiate. Hu and Rijkenberg (1998) illustrated progression of P. recondita f. sp. tritici. through all stages of development of various infection structures on the leaves of RL 6040 and RL6043 carrying leaf rust resistance genes Lr19 and Lr21 respectively and compared the ultrastructures with susceptible wheat cultivars. Considerable differences in infection structure development was not observed among the three wheat lines. Formation of two or three appressoria on one stoma, mature ellipsoidal SSV parallel to stomatal slit and formation of HMC were visible in all three wheat cultivars. Whereas in the present study, wheat isoline containing the resistance gene Lr28 showed no development of infection structures up to 168 hpi. A similar study was also conducted on common wheat by Bolton et al., (2008) where formation of SSVs occurred within 24 h whereas in the present study SSV was formed after 24 h i.e. at 72 hpi exhibiting a paramount difference in specific time points on the formation of various infection structures. Different wheat genotypes with their diverse genic contents might be attributed to play discrete roles.

The in-silico identification of flavonoid biosynthetic genes from SOLiD-SAGE libraries developed from susceptible and resistant wheat NILs with and without infection by P. triticina and their characterization undertaken in this study helped to a large extent in understanding the properties of the selected genes. Differential gene expression analysis of the initial (CHS) and middle stage (F3H) and late stage (ANS) genes distinctly indicated wheat plants responded to leaf rust infection in a dynamic yet coordinated manner. The study revealed the roles of flavonoid biosynthetic genes in defense mechanism during leaf rust in susceptible plants. This implicates that upregulation of the flavonoid biosynthetic genes, in the susceptible plants particularly during early stages (24–48 hpi) of infection might be involved in the production of flavonoids that acts as protective molecules and bolsters the defense mechanism. On the contrary, the resistance plants signify the protective role of Lr28 gene under leaf rust infection. It might be imperative that these genes encode regulatory proteins/peptides that are activated by a signal cascade of the flavonoid biosynthetic pathway and further branch into various pathways for the accumulation of several protective molecules in safeguarding the plant against pathogenesis.

Competing interest: The authors declare no conflict of interests. All authors read and approved the manuscript.

Author contributions: KM conceived and designed the experiments; MA performed the experiments and analyzed the data; BS supervised the work; SKJ preformed infectivity experiments; MK executed UHPLC-MS/MS; PKD interpreted LC-MS/MS data.

Acknowledgments: The authors sincerely acknowledge the Department of Bioengineering and Biotechnology for providing infrastructure to carry out the research and BTISNet SubDIC (BT/BI/04/065/04) for providing bioinformatics facilities. MA is also thankful to University Grants Commission of India (UGC) through Maulana Azad National Fellowship (MANF) [F1-17.1/2015-16/MANF-2015-17-JHA-58235/(SA-III)] for providing fellowship.

Author contributions:

KM conceived and designed the experiments; MA performed the experiments and analyzed the data; BS supervised the work; SKJ preformed infectivity experiments; MK executed UHPLC-MS/MS; PKD interpreted LC-MS/MS data.

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Reviewers agreed at journal
24 Sep, 2023
Reviewers invited by journal
29 Aug, 2023
Editor assigned by journal
26 May, 2023
First submitted to journal
24 May, 2023

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Identification and molecular characterization of flavonoid biosynthetic genes and their expression analysis in wheatgrass (Triticum aestivum L.) during leaf rust infection

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Abstract

Figures

Introduction

Materials and methods

Plant growth, pathogen inoculation and leaf sample collection

Estimation of total phenolic content

Estimation of total flavonoid content

Estimation of total monomeric anthocyanin content

Ultra-high-performance liquid chromatography LTQ-XL iontrap MS/MS analysis

Gene ontology annotation

Gene identification

Multiple sequence alignment and construction of phylogenetic tree

Gene expression analysis using quantitative Real-Time PCR

Specimen preparation and Scanning Electron Microscopy (SEM)

Statistical analysis

Results

Determination of Total Phenolic Content (TPC), Total Flavonoid Content (TFC) and anthocyanin content

Flavonoid profiling by LC-MS/MS in leaves of susceptible and resistant wheat NILs

Gene annotation

Expression profiling of flavonoid biosynthesis genes in wheat NILs in response to leaf rust

Scanning of uredinial infection development events at different time points using scanning electron microscope (SEM)

Discussion

Conclusion

Statements and Declarations

References

Supplementary Files

Status:

Version 1