Fig 1.
A. The defense MAPKs have been identified through transgenic analyses of all 32 members of the gene family [11]. RNA has then been isolated from the MAPK-OE defense-MAPK lines as well as their accompanying pRAP15 controls for RNA seq analyses [28]. B. In an associated analysis of the gene expression occurring within the syncytium (upper right panel), two different G. max genotypes that are capable of a defense response (G. max[Peking/PI 548402] and G. max[PI 88788]) have been analyzed. C. The RNA seq study has identified 309 genes that are induced in common between each of the MAPK-OE lines (MAPK-OE-all). D. The 1,787 syncytium-expressed gene list has been to be used to identify whether any of those genes are regulated by MAPKs (shown in C). E. A comparison of C and D gene lists has resulted in the identification of 71 genes that are in common between the MAPK-all and syncytium expressed gene analyses. F. Prior analyses have demonstrated the G. max secretion apparatus is important in defense to H. glycines parasitism [13, 14, 30]. The 71 candidate defense genes have been further narrowed down for the functional studies by conceptually translating their protein products to determine if any are predicted to be secreted proteins. The analyses has employed SignalP-5.0 leading to the identification of 8 (of the 71) genes that have a predicted secretion signal [36]. These 8 genes became the candidate defense genes that have been tested in transgenic studies presented here. Please see the Materials and Methods section for analysis details.
Table 1.
Detection call methodology as compared to the MAPK-OE-all induced genes, leading to the identification of 71 genes having measured expression in each analysis.
Fig 2.
The Gene Ontology analysis of the 309 MAPK-OE-all genes.
A. Induced genes GO analysis-biological process. B. Suppressed genes GO analysis-biological process. C. Induced genes GO analysis-molecular function. D. Suppressed genes GO analysis-molecular function. E. Induced genes GO analysis-cellular component. F. Suppressed genes GO analysis-cellular component. Gene Ontologies, specifically molecular function, have been retrieved from Phytozome, using the PhytoMine tool (https://phytozome.jgi.doe.gov/phytomine/begin.do). Graphs have been generated using Excel.
Fig 3.
The Gene Ontology analysis of the 71 MAPK-OE-all genes identified from the laser microdissected (LM) syncytium cell analyses and the MAPK-OE-all genes induced gene analyses.
The analyses included the 0 dpi (control), 3 and 6 dpi LM syncytia undergoing a defense response in G. max[Peking/PI 548402] and G. max[PI 88788]. A. GO analysis-biological process. B. GO analysis-molecular function. C. GO analysis-cellular component. Gene Ontologies, specifically molecular function, have been retrieved from Phytozome, using the PhytoMine tool (https://phytozome.jgi.doe.gov/phytomine/begin.do). Graphs have been generated using Excel.
Fig 4.
A qRT-PCR confirmation of the RNA seq gene expression data occurring for the 8 genes targeted for transgenic functional analyses that are MAPK-OE-all expressed, syncytium-expressed and are putatively secreted.
The target genes are a galactose mutarotase-like (GAL MUT, Glyma.19G020700), Pollen Ole e 1 allergen and extensin family protein (EXT, Glyma.13G178700); endomembrane protein 70 protein family (ENDO 70, Glyma.09G096700); O-Glycosyl hydrolases family 17 protein (HYDROL-17, Glyma.14G020000); glycosyl hydrolases family 32 protein (HYDROL-32, Glyma.13G349300); FASCICLIN-like arabinogalactan protein 17 precursor (AGP 17, Glyma.12G096300); peroxidase superfamily protein (PEROX, Glyma.01G171100); pathogenesis-related thaumatin superfamily protein (PR, Glyma.12G064300). A minimum cutoff is set at ± 1.5 fold. * Statistically significant. The p-values (p < 0.001) for the replicated qPCR analyses have been calculated through a Student’s t-test [33]. Please refer to Methods for analysis details. The qPCR analyses have been averaged for 3 independent replicates. MK = MAPK.
Fig 5.
Confirmation of effect the candidate defense gene expression cassette (OE or RNAi) has on the relative level of expression of the target gene.
The p-values (p < 0.001) for the replicated qPCR analyses have been calculated through a Student’s t-test [33]. Please refer to Methods for analysis details. The qPCR analyses have been averaged for 3 independent replicates.
Fig 6.
Functional transgenic analyses.
The analyses demonstrate that the genetic engineering of the putatively secreted candidate defense gene functions as expected in the G. max defense to H. glycines. The calculated FI for the OE and RNAi lines is presented as compared to the controls. The analyzed genes are a galactose mutarotase-like (GAL MUT, Glyma.19G020700), Pollen Ole e 1 allergen and extensin family protein (EXT, Glyma.13G178700); endomembrane protein 70 protein family (ENDO 70, Glyma.09G096700); O-Glycosyl hydrolases family 17 protein (HYDROL-17, Glyma.14G020000); glycosyl hydrolases family 32 protein (HYDROL-32, Glyma.13G349300); FASCICLIN-like arabinogalactan protein 17 precursor (AGP 17, Glyma.12G096300); peroxidase superfamily protein (PEROX, Glyma.01G171100); pathogenesis-related thaumatin superfamily protein (PR, Glyma.12G064300). * Statistically significant, p < 0.05 calculated by the Mann–Whitney–Wilcoxon (MWW) Rank-Sum Test [35]. The experimental error representing standard deviation is presented. The results are the average of three independently run biological replicates, all p values < 0.001.
Fig 7.
A. Example of the relatively low expression concentration of 8 candidate resistance genes in a naturally H. glycines-susceptible genotype (i.e. G. max[Williams 82/PI 518671]) shown with 10 H. glycines cysts. B. The H. glycines-susceptible genotype (i.e. G. max[Williams 82/PI 518671]), when engineered to overexpress one of the 8 candidate defense genes, results in a resistant reaction. The reaction is not exactly like a naturally occurring resistant reaction like that occurring in G. max[Peking/PI 548402] (like shown in C) as indicated by the presence of more cysts (n = 3) than the naturally occurring resistant reaction (n = 1, in C). The level of engineered resistance would depend on the potency and/or timing of the expression of the gene. C. 8 Candidate defense genes have been determined to be more highly expressed in the syncytium undergoing a defense reaction to H. glycines parasitism in a naturally H. glycines-resistant genotype (i.e. G. max[Peking/PI 548402]). D. The H. glycines-resistant genotype (i.e. G. max[Peking/PI 548402]), when engineered to undergo RNAi of one of the candidate defense genes, results in an engineered susceptible reaction. The reaction is not exactly like a naturally occurring susceptible reaction as found in G. max[Williams 82/PI 518671] (as shown in A) as indicated by fewer cysts (n = 7) than the naturally occurring susceptible reaction (n = 10, in A). The level of engineered susceptibility would depend on the importance of the gene to the defense reaction, its potency and/or the timing of its expression. The relative numbers of cysts are arbitrary for descriptive purposes.