Genome and transcriptome exploration reveals receptor-like kinases as potential resistance gene analogs against bacterial blight in pomegranate.

BACKGROUND: Pomegranate (Punica granatum L.) is a tropical fruit crop of pharma-nutritional importance. However, it faces farming challenges due to pests and diseases, particularly bacterial blight and wilt. Developing resistant cultivars is crucial for sustainable pomegranate cultivation, and understanding resistance's genetic basis is essential. METHODS AND RESULTS: We used an extensive resistance gene analogues (RGA) prediction tool to identify 958 RGAs, classified into Nucleotide Binding Site-leucine-rich repeat (NBS-LRR) proteins, receptor-like kinases (RLKs), receptor-like proteins (RLPs), Transmembrane coiled-coil (TM-CC), and nine non-canonical RGAs. RGAs were distributed across all eight chromosomes, with chromosome 02 containing the most RGAs (161), and chromosome 08 having the highest density (4.42 RGA/Mb). NBS-LRR genes were predominantly present on chromosomes 08 and 02, whereas RLKs and RLPs were primarily located on chromosomes 04 and 07. Gene ontology analysis revealed that 475 RGAs were associated with defence against various biotic stresses. Using RNAseq, we identified 120 differentially expressed RGAs, with RLKs (74) being prominent among the differentially expressed genes. CONCLUSION: The discovery of these RGAs is a significant step towards breeding pomegranates for pest and disease resistance. The differentially expressed RLKs hold promise for developing resistant cultivars against bacterial blight, thereby contributing to the sustainability of pomegranate cultivation.

Identification and validation of SSR markers for Xanthomonas axonopodis pv. punicae an incitant of bacterial blight of pomegranate.

This study reports genome wide characterization and development of first set of microsatellite markers through in silico analysis of eight sequenced Xanthomonas axonopodis pv. punicae strains available in the public database. SSR survey resulted in identification of ~ 4638 perfect SSRs, with mean marker frequency 901 SSRs/Mb and densitiy of 11,006 bp/Mb aross the eight genomes. Frequency distribution graphs revealed hexa-nucleotide repeats were more prominent fowllowed by tri-, tetra-, di- and penta-nucleotides in the analysed genomes. We desinged 2927 SSR primers that are specific to the strain LMG 859 and ePCR confirmed on seven other Xap genomes. This resulted in identification of 542 informative SSRs that are producing single amplicons, from which 66 primers were successfully validated through wet lab experiments on eight Xap isolates of pomegranate. Furthermore, utility of these SSRs were demostrated by analysing molecular diversity among 22 Xap isolates using 20 Xap_SSR primers. SSRs revealed moderate genetic diversity among Xap isolates (61%) and grouped 11 isolates that are repersenting six different states into one cluster. This proved the earlier evidence of wider spread of ST3 type Xap acoss India using Multi locus Sequence Typing (MLST) technique. In summary, Xap_SSR will serve as powerful genomics tools that would helps in monitoring of population dynamics, taxonomy, epidomology and quarantine aspects in bacterial blight pathogen through development of microsatellite based Multilocus Variable number of Tandem repeat analysis (MLVA) in future. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03209-z.

Long-Read Genome Sequence Resources of Xanthomonas citri pv. punicae Strain Bagalkot Causing Pomegranate Bacterial Blight.

Xanthomonas citri pv. punicae causing bacterial blight is a devastating disease of pomegranate in India and Pakistan. Most xanthomonads use the type III secretion system to inject transcription activator-like effector (TALE) proteins into the host cell. TALEs bind to the effector-binding elements in the promoter of host susceptibility genes, triggering disease development. PacBio single-molecule real-time long-read sequencing technology was used to identify the TALE-encoding genes, which is otherwise not possible using next-generation short-read sequencers. In all, 1.74 Gb of raw data containing 368,980 subreads, with an average read length of 4,724 bp and longest read length of 77,471, were generated. Subreads were assembled into 15 scaffolds generating approximately 5.4 Mb (348×) of genome. X. citri pv. punicae exhibited close lineage with X. citri pv. citri with 98.78% average nucleotide identity. Of the 4,263 protein-coding genes, 11 non-TALE type III effectors and 2 TALE-encoding genes were identified.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Reliable and early diagnosis of bacterial blight in pomegranate caused by Xanthomonas axonopodis pv. punicae using sensitive PCR techniques.

Bacterial blight caused by Xanthomonas axonopodis pv. punicae is a major disease of pomegranate. Bacterial blight drastically reduces the yield and quality of fruits, which are critical for pomegranate production. Precise and early diagnosis of bacterial blight is crucial for active surveillance and effective management of the disease. Symptoms based disease diagnostic methods are labor-intensive, time-consuming and may not detect disease on asymptomatic plants. DNA-based disease diagnostics using polymerase chain reaction (PCR) are reliable, precise, accurate and quick. PCR coupled with agarose gel electrophoresis (PCR-AGE), PCR coupled with capillary electrophoresis (PCR-CE) and real-time PCR (qPCR) were applied for the early and accurate diagnosis of bacterial blight in pomegranate. PCR-CE and qPCR were capable of diagnosing bacterial blight 6 to 10 days before symptom appearance, with detection limits of 100 fg and 10 fg of bacterial DNA respectively. However, conventional PCR-AGE detected pathogen at the onset of disease symptoms with a detection limit of 10 pg of bacterial DNA. qPCR detected bacterial blight in orchards that did not show any disease symptoms. Our data demonstrate that qPCR is more sensitive than other PCR methods along with being reliable for early diagnosis.

Metabolomics deciphers the host resistance mechanisms in wheat cultivar Sumai-3, against trichothecene producing and non-producing isolates of Fusarium graminearum.

Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum, reduces grain yield and contaminates grains with trichothecene mycotoxins. Host resistance to FHB is quantitatively inherited and more than 100 QTLs have been mapped, but the host resistance mechanisms are poorly understood. Non-targeted metabolic profiling was applied to elucidate the host resistance mechanisms to FHB spread through rachis of wheat cultivar Sumai-3 against both trichothecene producing and non-producing isolates of Fusarium graminearum. The accumulation of deoxynivalenol (DON) in Sumai-3 was low, however the resistance to spread was not due to its detoxification into DON-3-O-glucoside (D3G), as the proportion of total DON converted to D3G in the resistant was not significantly different from that in the susceptible cultivar Roblin. Instead, the resistance was considered to be due to the accumulation of resistance related (RR) metabolites belonging to the phenylpropanoid pathway that reduced pathogen advancement through increased host cell wall thickening and also reduced pathogen growth due to antifungal and/or antioxidant properties which, in turn, reduced subsequent trichothecene biosynthesis. The RR phenylpropanoids accumulated in Sumai-3 were mainly the preformed syringyl rich monolignols and their glucosides, which are precursors of lignin biosynthesis, as well as antimicrobial flavonoids. The resistant cultivar Sumai-3 inoculated with trichothecene producing F. graminearum not only accumulated less RR metabolites but also the abundance of many RR metabolites was lesser than in the trichothecene non-producing F. graminearum. This implies repression of host resistance mechanisms by trichothecenes/DON, which is a protein biosynthesis inhibitor. Enhancement of resistance in wheat against FHB can be exploited through stacking of candidate phenylpropanoid pathway genes.

Metabolo-proteomics to discover plant biotic stress resistance genes.

Plants continuously encounter various environmental stresses and use qualitative and quantitative measures to resist pathogen attack. Qualitative stress responses, based on monogenic inheritance, have been elucidated and successfully used in plant improvement. By contrast, quantitative stress responses remain largely unexplored in plant breeding, due to complex polygenic inheritance, although hundreds of quantitative trait loci for resistance have been identified. Recent advances in metabolomic and proteomic technologies now offer opportunities to overcome the hurdle of polygenic inheritance and identify candidate genes for use in plant breeding, thus improving the global food security. In this review, we describe a conceptual background to the plant-pathogen relationship and propose ten heuristic steps streamlining the application of metabolo-proteomics to improve plant resistance to biotic stress.

Integrated metabolo-proteomic approach to decipher the mechanisms by which wheat QTL (Fhb1) contributes to resistance against Fusarium graminearum.

BACKGROUND: Resistance in plants to pathogen attack can be qualitative or quantitative. For the latter, hundreds of quantitative trait loci (QTLs) have been identified, but the mechanisms of resistance are largely unknown. Integrated non-target metabolomics and proteomics, using high resolution hybrid mass spectrometry, were applied to identify the mechanisms of resistance governed by the fusarium head blight resistance locus, Fhb1, in the near isogenic lines derived from wheat genotype Nyubai. FINDINGS: The metabolomic and proteomic profiles were compared between the near isogenic lines (NIL) with resistant and susceptible alleles of Fhb1 upon F. graminearum or mock-inoculation. The resistance-related metabolites and proteins identified were mapped to metabolic pathways. Metabolites of the shunt phenylpropanoid pathway such as hydroxycinnamic acid amides, phenolic glucosides and flavonoids were induced only in the resistant NIL, or induced at higher abundances in resistant than in susceptible NIL, following pathogen inoculation. The identities of these metabolites were confirmed, with fragmentation patterns, using the high resolution LC-LTQ-Orbitrap. Concurrently, the enzymes of phenylpropanoid biosynthesis such as cinnamyl alcohol dehydrogenase, caffeoyl-CoA O-methyltransferase, caffeic acid O-methyltransferase, flavonoid O-methyltransferase, agmatine coumaroyltransferase and peroxidase were also up-regulated. Increased cell wall thickening due to deposition of hydroxycinnamic acid amides and flavonoids was confirmed by histo-chemical localization of the metabolites using confocal microscopy. CONCLUSION: The present study demonstrates that the resistance in Fhb1 derived from the wheat genotype Nyubai is mainly associated with cell wall thickening due to deposition of hydroxycinnamic acid amides, phenolic glucosides and flavonoids, but not with the conversion of deoxynivalenol to less toxic deoxynivalenol 3-O-glucoside.