| Rice?Oryza sativa L.?is among the important staple food crops,fulfilling food requirements of about half of the world's human population.The global human population is expected to increase to around 9 billion at 2050;and thus,to feed this increased population,enhancement in the yield of rice is extremely critical.Unfortunately,rice blast disease caused by the hemibiotrophic fungal pathogen,Magnaporthe oryzae,leads to approximately 10-30%damage to the rice harvest world-wide per annum,which,therefore,remains a major threat to food security globally.Rice blast disease is typically controlled by the use of different fungicides such as tricyclazole,probenazole,azoxystrobin,propiconazole and iprobenfos.In addition to problems with the efficacy of these fungicides,there has been a growing concern over the inappropriate use of chemical fungicides,which can lead to potentially hazardous health and environmental issues.The use of crop varieties with“race-specific”resistance is another reliable method of disease management;however,in the case of rice blast disease,resistance is not durable,only lasting about two or three years,due to the occurrence of new bio-types of M.oryzae that constantly overcome host defense.Thus,there is a critical need to improve rice immunity against M.oryzae using alternative approaches that have the potential to provide a more durable resistance to rice blast disease.Of note,to tackle diverse array of biotic and abiotic stresses,plants not only activate basal defense responses,including defense hormone production,but also rely on rhizomicrobiomes for survival and fitness.The involvement of rhizomicrobiomes in the host nutrient acquisition and environmental stress resilience have made them ideal target to enhance agricultural gains in an eco-sustainable way.Fascinatingly,specific phytohormone-biosynthesis and–signaling pathways not only sever as plant growth regulators and defense molecules,but also play active roles in recruiting specific rhizomicrobiome.Therefore,uncovering the role of phytohormones in defense and determining the rhizomicrobiome as well as its relevant mechanisms will provide new avenues to enhance crop yield in an eco-friendly way.Strigolactones?SLs?are a group of phytohormones that paly critical roles in the regulation of plant developmental and physiological processes.Besides,emerging evidence suggests that SLs also promote specific plant biotic and abiotic stress resistance.In addition to this,SLs also act as rhizosphere signaling molecules;thereby positively regulating the symbiotic interaction among the host plant and arbuscular mycorrhizal fungi.Notably,recent studies implicated the involvement of SLs in determining rhizomicrobiome composition.These finding suggest the potential regulatory roles of SLs during plant-microbe interactions.However,to our knowledge,the possible regulatory roles and underlying molecular mechanisms of SL-biosynthesis and–signaling in rice blast defense and shaping rhizomicrobiome remain unexplored.Thus,in this research study we investigated the possible regulatory roles and underlying molecular mechanisms of SL-biosynthesis and–signaling in rice blast defense and shaping rhizomicrobiome.In this first line of investigation,we explored the contribution of SL-biosynthesis and-signaling in defense against the rice blast by comparative phenotypic analysis of the rice mutants that are impaired in either SL biosynthesis or signaling and their corresponding wild-type?WT;O sativa L.cv.Shiokari?rice plants during M.oryzae infection.Results indicate that mutations in either SL-biosynthetic?dwarf17,d17?or–signaling?d14?led to increased susceptibility towards M.oryzae.This clearly suggests that SL-biosynthesis and–signaling pathways are involved in resistance to rice blast disease.To explore SL-regulated downstream genes/pathways involved in resistance to rice blast fungus,RNA-seq-based comparative leaf transcriptomic profiling of the d14 mutants and WT rice plants with and without M.oryzae infection were conducted.Next,we took advantage of sophisticated–omics tool known as MapMan and annotated our transcriptome data to biotic stress overview?pathway?.MapMan-based biotic stress overview of the transcriptome datasets revealed that substantial number of defense-associated genes,including cell wall-,ethylene?ET?-,hydrogen peroxide synthesis?H2O2?-related genes were remarkably suppressed in the leaves of d14 mutants with respect to the WT plants,during M.oryzae infection.In addition,the Kyoto Encyclopedia of Genes and Genomes?KEGG?pathway annotation analysis indicated that various metabolic pathways mostly related to carbohydrate/sugar metabolism,including‘starch and sucrose metabolism',‘pentose phosphate pathway',‘glyoxylate and dicarboxylate metabolism',‘photosynthesis'and‘carbon fixation in photosynthetic organisms'were suppressed at significant levels in the d14 mutants compared with WT plants,during rice blast infection.The transcriptome data results were further validated by analyzing the expression patterns of eight genes related to ET biosynthesis-,H2O2 biosynthesis-,cell wall synthesis-and sugar metabolism-related-pathways,as well as seven randomly selected genes using quantitative reverse transcription PCR?qRT-PCR?.In general,qRT-PCR showed that the genes analyzed showed almost the same trends in their expression as that of RNA-seq data,which confirms the accuracy and reliability of the transcriptome data.Additionally,we also carried out histochemical detection of H2O2 and soluble sugar measurement in the leaves of rice blast-infected d17,d14 and WT plants.In support of transcriptome data results,here,the biochemical findings revealed that accumulation of H2O2?evidenced by DAB staining intensities?and soluble sugar content were remarkably reduced in the leaves of both mutants?d17 and d14?relative to WT,further pointing towards the role of SL-dependent promotion of H2O2 and sugar content,especially during biotic stress.Moreover,we also investigated the possible direct impact of synthetic SL?GR5?on growth of rice blast fungus.No significant impact of SLs on rice blast growth was observed,which further supported the in-planta SL-mediated defense mechanisms rather that its direct influence on rice blast growth.Altogether,these findings present evidence that SL-biosynthesis and–signaling promote defense against rice blast infection through involvement in the induction of cell wall-,ET-,H2O2-and sugar biosynthesis-related genes/pathways.The findings of this study proposing that SLs may serve an additional?novel?target for enhancement of rice defense against M.oryzae.In the next line of observation,to address the question of whether and how SL-biosynthesis and–signaling determine the rhizomicrobiomes in rice,we analyzed and compared the rhizomicrobiome?which are based on 16S rRNA and internal transcribed spacer amplicon data?of rice d17 and d14 mutants with the relevant WT rice.Compared to WT,higher bacterial richness?evidenced by the operational taxonomic unit richness?and lower fungal diversity?evidenced by the Shannon index?were observed in the rhizosphere of both SL-biosynthetic d17and–signaling d14 mutant lines.In addition,d14 showed lower bacterial diversity?evidenced by Simpson index?at a significant level,while it was slightly significant in d17 with respect to WT.Both principal coordinate analysis and permutational multivariate analysis of variance based on Bray-Curtis dissimilarities of the filtered operational taxonomic units revealed significant differences in the root-associated microbiome of d17 and d14 compared with the WT.Interestingly,remarkable differences in the composition of a large number of bacterial communities,whereas few fungal communities were detected in the rhizosphere both d17 and d14 mutants with respect to the WT,proposing that rice SL-biosynthesis and–signaling exert greater impact on structuring bacterial communities rather than fungal communities.Notably,16S rRNA sequencing indicated that the relative abundance of certain beneficial bacterial taxa,including that of Nitrosomonadaceae and Rhodanobacter,were significantly decreased in both mutant genotypes relative to the WT.Members of Nitrosomonadaceae are likely to promote nitrification,while species within Rhodanobacter have the capacity to act as potential bio-control agents.This finding,presents indirect evidence that SL-biosynthesis and–signaling may mediate various biotic and biotic stresses,probably,through the positive regulation of the respective bacterial composition.To understand whether SL-dependent metabolic pathways shape the rhizomicrobiome,a correlation network analysis was conducted among the metabolic pathway-related genes?which are based on transcriptome data?and the rhizomicrobiome of rice.Correlation network analysis between SL-dependent metabolic pathway-associated genes and rhizomicrobiome proposed a role for SL-dependent metabolic pathways in shaping rhizomicrobiome composition.In this regard,specific SL-dependent metabolic pathways,including‘Phenylalanine metabolism',‘Phenylpropanoid biosynthesis',‘Plant hormone signal transduction'‘Peroxisome',‘Glyoxylate and dicarboxylate metabolism',‘Galactose metabolism'and‘Alanine,aspartate,and glutamate metabolism'pathways and rhizomicrobiome taxa enriched in WT were positively correlated.These findings suggest that SL-biosynthesis and–signaling play a key role in determining the rhizomicrobiome composition in rice,directly or indirectly through the mediation of distinct metabolic pathways.In conclusion,these results present evidence that SL-biosynthesis and–signaling not only positively regulate rice defense against M.oryzae,but also play a key role in shaping the rhizomicrobiome,particularly root-associated bacterial communities,possibly through the mediation of distinct metabolic pathways.Thus,based on our findings,we propose that manipulation of SL biosynthesis and/or signaling pathways via transgenic approaches may provide a novel strategy to enhance defense mechanisms of rice against M.oryzae,and may also help to recruit and/or increase the relative abundance of the desired beneficial rhizomicrobiome,which may assist in stress resilience of rice and perhaps other crops.In turn,this will enable us to achieve the ultimate goal of ensuring sustainable food security. |
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