Integrated Omic Approaches Reveal Molecular Mechanisms of Tolerance during Soybean and Meloidogyne incognita Interactions

Abstract

The root-knot nematode (RKN), Meloidogyne incognita, is a devastating soybean pathogen worldwide. The use of resistant cultivars is the most effective method to prevent economic losses caused by RKNs. To elucidate the mechanisms involved in resistance to RKN, we determined the proteome and transcriptome profiles from roots of susceptible (BRS133) and highly tolerant (PI 595099) Glycine max genotypes 4, 12, and 30 days after RKN infestation. After in silico analysis, we described major defense molecules and mechanisms considered constitutive responses to nematode infestation, such as mTOR, PI3K-Akt, relaxin, and thermogenesis. The integrated data allowed us to identify protein families and metabolic pathways exclusively regulated in tolerant soybean genotypes. Among them, we highlighted the phenylpropanoid pathway as an early, robust, and systemic defense process capable of controlling M. incognita reproduction. Associated with this metabolic pathway, 29 differentially expressed genes encoding 11 different enzymes were identified, mainly from the flavonoid and derivative pathways. Based on differential expression in transcriptomic and proteomic data, as well as in the expression profile by RT-qPCR, and previous studies, we selected and overexpressed the GmPR10 gene in transgenic tobacco to assess its protective effect against M. incognita. Transgenic plants of the T₂ generation showed up to 58% reduction in the M. incognita reproduction factor. Finally, data suggest that GmPR10 overexpression can be effective against the plant parasitic nematode M. incognita, but its mechanism of action remains unclear. These findings will help develop new engineered soybean genotypes with higher performance in response to RKN infections.

Keywords: differential expression; phenylpropanoids; proteome; root-knot nematode; transcriptome.

Figure 1

Morphological analysis showing the interaction of soybean roots from two contrasting soybean genotypes: BRS 133 (a–e) and PI 595099 (f–j), inoculated with the nematode M. incognita. In (a,f), root tips inoculated with the nematode are presented 4 days after inoculation (DAI). The penetration coefficient (PEf) is similar for both genotypes. In (b,g), the observed differences in morphology from the galls are shown (BRS 133 is elongated while PI 595099 is ovoid). Comparing the galls or root tips isolated from BRS 133 (c–e) and PI 595099 (h–j) evidence that the time course of M. incognita infection in both genotypes is similar. Only a slight delay in the development of galls and a smaller cytoplasmic volume in the giant cells were observed in PI 595099 samples. GC—giant cell; J2—juvenile phase 2 of M. incognita development; Mi—M. incognita; PEf—penetration efficiency; RF—reproduction factor.

Figure 2

Enrichment of Gene Ontology (GO) terms in the transcriptome of G. max infected with M. incognita. The susceptible BRS 133 and the tolerant PI 595099 genotypes are represented at the top of the bubble chart, followed by the number of days after the infection (DAI) with M. incognita. The GO terms are listed on the left (y-axis). Only GOs with statistical significance (false discovery rate, FDR > 0.05) are included.

Figure 3

Enrichment of metabolic pathways from Kyoto Encyclopedia of Genes and Genomes (KEGG) database in the transcriptome and proteome of G. max infected with M. incognita. The susceptible BRS 133 and the tolerant PI 595099 genotypes are represented at the top of the bubble chart, followed by the number of days after the infection (DAI) with M. incognita. Major metabolic pathways are listed on the right (y-axis). Minor metabolic pathways are listed on the left (y-axis). Only pathways with statistical significance (false discovery rate, FDR > 0.05) are included. No enriched terms were Identified to tolerant PI 595099 genotype at 4 DAI.

Figure 4

Phenylpropanoid-derived metabolic pathways associated with soybean tolerance to M. incognita. According to the proteome and transcriptome analyses, the tolerance to root-knot nematode observed in the soybean genotype PI 595099 might be associated with a super production of secondary metabolites derived from the phenylpropanoids pathway, which directly affect nematodes parasitism. (a) Main branch of the phenylpropanoids pathway that converts the aromatic amino acid phenylalanine into p-Coumaric Acid and 4-Coumaroil-CoA. Phenylalanine is converted into cinnamic acid by the enzyme Phenylalanine Ammonia-Lyase (PAL) and subsequently into p-Coumaric Acid by the Cinnamate-4-Hydroxilase (C4H) and upregulated in the tolerant genotype. The p-Coumaric Acid is finally converted into 4-Coumaroil-CoA by the 4-Coumarate-CoA-Ligase, and also upregulated in the highly tolerant genotype. p-Coumaric Acid and 4-Coumaroil-CoA are the molecular backbones for the aromatic-derivative metabolic routes. (b) Pterocarpans-derived pathway, converging to glyceollin biosynthesis. The initial and final steps, comprising the reaction catalyzed by Chalcone Synthase (CHS) and Isoflavone Reductase (IFR), are hyperactive in the tolerant genotype. (c) Flavanone biosynthesis pathway, converging to naringenin production. The initial step of this derivative route is shared between flavanones and pterocarpans through the reaction catalyzed by the CHS. (d) Anthocyanins-derived route. Flavonoid-3′-Hydroxylase and Leucoanthocyanidin Dioxygenase (F3′H and LDOX), the main enzymes involved in the synthesis of the active anthocyanins pelargonidin and cyanidin, are upregulated in the tolerant genotype. (e) Lignin biosynthesis relies on the polymerization of aromatic alcohols derived from phenylalanine and tyrosine. A step of this pathway, which converts p-Coumaryl Alcohol into Coniferyl Alcohol and Sinapyl Alcohol (catalyzed by the enzyme Caffeoyl-CoA-3-o-Methyltransferase—CCoAMT) is upregulated in PI 595099 genotype. The final steps of lignin polymerization, catalyzed by cyclic reaction of Laccases (LCC) and Peroxidases (POX), are also upregulated in PI 595099. Light blue squares: no changes detected in expression levels. Dark blue squares: upregulated enzymes. 4CL—4-Coumarate-CoA-Ligase; C3′H—p-Coumaroyl-5-O-Shikimate 3′-Hydroxylase; C4H—Cinnamate-4-Hydroxilase; CAD—Cinnamyl Alcohol Dehydrogenase; CCoAMT—Caffeoyl-CoA-o-Methyltransferase; CCR—Cinnamoyl-CoA Reductase; CHI—Chalcone Isomerase; CHS—Chalcone Synthase; DFR—Dihydroflavonol 4-Reductase; F3′H—Flavonoid 3′-Hydroxylase; HCT—Hydroxycinnamoyl-CoA: Shikimate Hydroxycinnamoyl Transferase; I2′H—Isoflavone-2′-Hydroxylase; IFD—2-Hydroxy-Isoflavone Dehydrase; IFR—Isoflavone Reductase; IFS—2-Hydroxy-Isoflavone Synthase; LCC—Laccase; LDOX—Leucoanthocyanidin Dioxygenase; PAL—Phenylalanine Ammonia-Lyase; POX—Peroxidase.

Figure 5

Distribution of differentially expressed genes encoding transcription factors (TFs) in the transcriptomes of genotypes BRS 133 and PI 595099 infected with M. incognita. In the heatmap are clusters of the following families of TFs: basic leucine zipper domain (bZIP), GATA family (GATA), GRAS-domain (GRAS), heat shock factors (HSFs), DNA-binding MADS domain (MADS-box), KNOX/ELK homeobox (KNOX/ELK), DNA-binding domain MYB domain (MYBs), plant AT-rich sequence- and zinc-binding (PLATZ), DNA-binding domain WRKY domain (WRKY), basic helix-loop-helix TFs (bHLH), homeobox protein BEL1 (BEL1), c2h2/c2hc zinc fingers (C2H2/C2HC ZF), apetala 2 (AP2), DNA-binding domain NAC domain (NAC) and growth regulating factors (GRFs). Numbers shown in parentheses after the TF families correspond to the number of DEGs. The susceptible genotype BRS 133 and the highly tolerant genotype PI 595099 are represented at the bottom of the chart. Column clusters were generated based on Euclidean distances. Values of fold-change (FC) for each genotype are given in comparison to the time zero [0 days after inoculation (DAI) with M. incognita] of the corresponding genotype.

Figure 6

Validation of differentially expressed genes profile. The graphs show the RT–qPCR relative expression (2^ΔΔCt) of 20 candidate genes selected after comparison of transcriptome and proteome data from two soybean genotypes (BRS 133 and PI 595099) inoculated (4, 12, and 30 days after inoculation—DAI) and not inoculated (0 DAI) with the nematode M. incognita. (a) acid phosphatase-related (GmAPHO; Glyma.08G200100.1); (b) alcohol dehydrogenase related (GmALCD; Glyma.04G240800.1); (c) annexin D1-related (GmAND1; Glyma.13G088700.1); (d) auxin associated protein (GmAUXA; Glyma.13G237000.1); (e) basic blue protein (GmBaBl; Glyma.08G128100.1); (f) pathogenesis-related protein Bet v-1 family (GmBetV or GmPR10; Glyma.17G030400.1); (g) β-amylase 5-related (GmBAM5; Glyma.06G301500.1); (h) calreticulin and calnexin (GmCALN; Glyma.10G147600.1); (i) catechol oxidase; tyrosinase (GmTYR; Glyma.15G071200.1); (j) cytochrome B5 isoform A (GmCYB5; Glyma.03G259600.1); (k) DR4 protein-related (GmDR4R; Glyma.09G155500.1); (l) 2-methylene-furan-3-one reductase; enone oxidoreductase (GmENOX; Glyma.19G008500.1); (m) ferritin heavy chain (GmFTH1; Glyma.01G124500.1); (n) glutathione S-transferase (GmGST; Glyma.18G190300.1); (o) not annotated 1 (GmNOA1; Glyma.01G018000.1); (p) not annotated 2 (GmNOA2; Glyma.06G056000.1); (q) not annotated 3 (GmNOA3; Glyma.19G114700.1); (r) trypsin and protease inhibitor (GmTRPI; Glyma.15G211500.1); (s) remorin, N-terminal region (GmREMN; Glyma.09G139200.1); and (t) universal stress protein family (GmUSP; Glyma.04G107900.1). The asterisks represent statistical significance in relation to the control sample (not inoculated) (t test, Bonferroni corrected; p ≤ 0.05).

Figure 7

GmPR10 overexpression in transgenic N. tabacum. (a) schematic representation of the transformation cassette cloned into the pPZP vector. The GmPR10 and eGFP (a selective marker) genes are both expressed through the CaMV35S promoter. Three independent transformation events were selected based on hygromycin resistance and eGFP fluorescence at 488 nm. (b1–e1) bright-field images of Ev11.1, Ev20.1, Ev23.2, and non-transformed wild-type (WT) tobacco plants and (b2–e1) images of eGFP fluorescence in the same plants. The GmPR10 and eGFP overexpression was evaluated by RT–qPCR as observed in (f). At 60 days after inoculation (DAI), the following parameters were analyzed in soybean-M. incognita bioassays: (g) galls per gram of root; (h) eggs per gram of root; and (i) M. incognita reproduction factor. The letters A/a/b presented in (f–i) correspond to groups of ANOVA statistical analysis.

Figure 8

Histological analysis of M. incognita-induced galls in transgenic Nicotiana tabacum overexpressing the soybean GmPR10 gene. At least five biological replicates from each line were infected with M. incognita, and gall and nematode morphology were evaluated 60 days after inoculation (DAI). (a1,a2) non-transformed wild-type (WT); (b1,b2) E11.1, (c1,c2) E20.1 and E23.2 (d1,d2). All sectioned galls were stained with toluidine blue. Gall from WT control presented multiple giant cells filled with dense cytoplasm and contained large nuclei. In contrast, galls from transgenic lines showed giant cells with few cytoplasm contents in E11.1 and E23.2 lines and, additionally, E20.1 line showed nematode with altered morphology and giant cells, which presented thinner cell walls. In this way, the analysis demonstrated that GmPR10 overexpression showed a direct effect on feeding site ontogeny. (a1–d1) zoom 1×. (a1–d2) zoom 10×. Legend: (*) giant cell; (Mi) M. incognita. Scale bars: 50 µm (red) and 2.5 µm (black).