An integrated RNAseq-1H NMR metabolomics approach to understand soybean primary metabolism regulation in response to Rhizoctonia foliar blight disease

Abstract

Background Rhizoctonia solani AG1-IA is a devastating phytopathogen causing Rhizoctonia foliar blight (RFB) of soybean worldwide with yield losses reaching 60%. Plant defense mechanisms are complex and information from different metabolic pathways is required to thoroughly understand plant defense regulation and function. Combining information from different “omics” levels such as transcriptomics, metabolomics, and proteomics is required to gain insights into plant metabolism and its regulation. As such, we studied fluctuations in soybean metabolism in response to R. solani infection at early and late disease stages using an integrated transcriptomics-metabolomics approach, focusing on the regulation of soybean primary metabolism and oxidative stress tolerance.ResultsTranscriptomics (RNAseq) and metabolomics (1H NMR) data were analyzed individually and by integration using bidirectional orthogonal projections to latent structures (O2PLS) to reveal possible links between the metabolome and transcriptome during early and late infection stages. O2PLS analysis detected 516 significant transcripts, double that reported in the univariate analysis, and more significant metabolites than detected in partial least squares discriminant analysis. Strong separation of treatments based on integration of the metabolomes and transcriptomes of the analyzed soybean leaves was revealed, similar trends as those seen in analyses done on individual datasets, validating the integration method being applied. Strong fluctuations of soybean primary metabolism occurred in glycolysis, the TCA cycle, photosynthesis and photosynthates in response to R. solani infection. Data were validated using quantitative real-time PCR on a set of specific markers as well as randomly selected genes. Significant increases in transcript and metabolite levels involved in redox reactions and ROS signaling, such as peroxidases, thiamine, tocopherol, proline, L-alanine and GABA were also recorded. Levels of ethanol increased 24 h post-infection in soybean leaves, and alcohol dehydrogenase (ADH) loss-of-function mutants of Arabidopsis thaliana had higher necrosis than wild type plants.ConclusionsAs a proof-of-concept, this study offers novel insights into the biological correlations and identification of candidate genes and metabolites that can be used in soybean breeding for resistance to R. solani AG1-IA infection. Additionally, these findings imply that alcohol and its associated gene product ADH may have important roles in plant resistance to R. solani AG1-IA causing foliar blight.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-017-1020-8) contains supplementary material, which is available to authorized users.

γ‐Aminobutyric acid and related amino acids in plant immune responses: Emerging mechanisms of action

“…Endurance‐based strategies are postulated to be effective against necrotrophs; however, they require great amounts of metabolic energy. Consistently, a strong depletion of Glu and GABA levels was recorded in soybean leaves infected with the necrotroph Rhizoctonia solani (Copley et al, ). Endurance strategies would likely need to be supplied with substrates coming from distal, healthy areas of the plant (Seifi, Van Bockhaven, et al, ).…”

“…Moreover, GAD is known to be activated following cytosol acidification (Shelp et al, ; Snedden et al, ). In fact, recognition of pathogens such as bacteria and fungi was proven to trigger GABA accumulation in several plant species, such as bean ( Phaseolus vulgaris L.), tomato ( Solanum lycopersicon L.) and soybean ( Glycine max L.) (Copley, Aliferis, Kliebenstein, & Jabaji, ; O'Leary et al, ; Wang et al, ) (Table ). The accumulated GABA is presumed to be transported to the mitochondria, where it undergoes deamination via GABA transaminase (GABAT) to succinic semialdehyde (SSA) (Shelp & Zarei, ).…”

The transcriptional landscape of Rhizoctonia solani AG1-IA during infection of soybean as defined by RNA-seq

“…The use of RNA-seq has provided substantial insights into plant-pathogen interactions; however, only recently have studies begun to examine the global transcriptome responses of phytopathogens such as Rhizoctonia solani while infecting host plants [ 12 , 13 ]. Although RNA-seq can provide information on both the host and pathogen, previous studies have reported that such studies provide few reads and therefore little information on the pathogen responses [ 41 , 44 , 45 ]. As such, a method using nitrocellulose membranes to obtain an in-depth and thus highly informative study of R .…”

“…Transcript levels were quantified in three biological replicates per treatment and significant differences determined using analysis of variance (ANOVA) and student’s t test at the 95% significance level using R statistics software. Fold change differences ≤ -1.5 or ≥ 1.5 with P <0.05 were used for qRT-PCR rather than ≤ -3 or ≥ 3 due to the differences in methods of normalization between RNA-seq and qRT-PCR [ 40 , 41 ].…”

Performance of methods that separate common and distinct variation in multiple data blocks

“…OnPLS is the multiblock extension of the O2PLS algorithm, and the method is often used for integrating –omics data . O2PLS is based on analysis of the covariance matrix of two blocks, .…”