Effects of sugarcane aphid herbivory on transcriptional responses of resistant and susceptible sorghum

- Background: Sugarcane aphid (Melanaphis sacchari) outbreaks in sorghum that were first reported in 2013 are now the most significant threat to this crop in all major sorghum production areas in the U.S.
- Further, gene ontology and pathway analysis indicated a suite of transcriptional changes in the resistant genotype that were weak or absent in the susceptible sorghum.
- sacchari up-regulated several genes involved in defense, which was particularly evident in the 2-wk plants that showed the most robust transcriptional responses..
- These transcriptional changes in the younger resistant sorghum were characterized by induction of hormone-signaling pathways, pathways coding for secondary metabolites, glutathion metabolism, and plant-pathogen interaction..
- These transcriptional responses were reflected in the aphid population growth, which was significantly faster in the susceptible and older sorghum than in the resistant and younger plants..
- Further, it appears that the younger resistant sorghum was able to mount a robust defense response following aphid herbivory, which was much weaker in the older sorghum.
- Invasive pests pose unique challenges to crop protec- tion, which was recently exemplified by an invasion of sugarcane aphid, Melanaphis sacchari Zehtner (Hemip- tera: Aphididae), a new invasive pest threatening sor- ghum production in the U.S.
- While the species has been present in the U.S.
- in sugarcane for over a cen- tury, an invasion of a new haplotype [21] absent in the country prior to the outbreaks is thought to be respon- sible for the eruptive population dynamics of M.
- sacchari is generally low [27, 28], and sugarcane aphids colonizing sorghum in the U.S.
- Therefore, host plant resistance has been successfully deployed to alleviate the impact of sugarcane aphids on sorghum and has proven to be an effective suppression tactic in the field [30–32].
- For example, Szczepaniec [32] reported that host plant resistance in the resistant genotype was the most effective factor in population dy- namics of M.
- Further, it has been demon- strated in the field that populations of these aphids can.
- Sugarcane aphids colonize sorghum in vegetative stages in the southern U.S., where they can overwinter on non-crop vegetation [23], while wind-aided migration is responsible for aphid colonization of sorghum in reproductive stages in the northern states (e.g., High Plains) [32].
- We hypothesized that expression of genes involved in defense pathways will be greater in the resistant and in younger sorghum than in the sus- ceptible and in older sorghum and that the aphid popu- lation growth will reflect these differences..
- Of the 5955 DEGs in the 2-wk resist- ant genotype exposed to aphid herbivory, 54.9% were up-regulated and 45% were down-regulated.
- were down-regulated in the 2-wk susceptible sorghum in response to the aphid (Additional files 2 and 3).
- Fewer responses were noted in the 6-wk sorghum.
- The overlaps between different sets of DEGs repre- senting changes in the 2- and 6-wk susceptible and re- sistant genotypes in response to aphid herbivory were further illustrated in Venn diagrams (Fig.
- In the 2-wk sorghum, 2430 DEGs were shared between both geno- types in response to aphid herbivory, while 3525 and 986 DEGs were only expressed in the resistant and sus- ceptible genotypes, respectively.
- The higher numbers of DEGs in the younger resistant genotype in response.
- Aphid herbivory affected a higher number of significant GO terms in the 2-wk plants than in the 6-wk plants re- gardless of genotype (Fig.
- In the resistant 2-wk geno- type, up-regulated genes associated with metabolic process, biosynthetic process, macromolecule process, and oxidation reduction were included in the enriched biological process GO terms.
- was also the prominent enriched GO term in the mo- lecular function category in the 2-wk resistant genotype (Fig.
- Similarly, we noted enrichment in polysacchar- ide biosynthesis, carbohydrate biosynthesis, metabolic process, and oxidation reduction GO terms in up- regulated genes in the 2-wk susceptible genotype (Fig..
- The number of enriched GO terms in both up- and down-regulated genes was higher in the 6-wk resistant genotype (Fig.
- 3c) than in the 6-wk susceptible genotype (Fig.
- The majority of the down-regulated genes were enriched in several nucleotide binding GO terms, and it was noteworthy that “oxidoreductase activity” was the only GO term that was down-regulated in the 6-wk susceptible genotype (Fig.
- was up-regulated in both 2- and 6-wk resistant genotype and in the 2-wk susceptible genotype (Fig.
- Our outcomes corroborate these studies and sug- gest that oxidative responses that are elicited in the re- sistant genotype are may be the key components of sorghum resistance to sugarcane aphids..
- Specifically, 3270 genes that mapped to 24 pathways were up-regulated in the 2-wk resistant genotype in response to aphid herbivory, while 1275 up-regulated genes from 10 pathways were elicited in the 2-wk.
- Fewer pathways were enriched in the 6-wk old sorghum..
- In general, we found that a significantly higher number of pathways were up regulated rather than down- regulated in the 2-wk old plants regardless of the genotype (Fig.
- It is noteworthy that in our study plant hor- mone signal transduction and glutathione metabolism pathways, both shown previously to be highly relevant to plant defense were only enriched in the 2-wk resistant genotype along with a plant-pathogen interaction pathway (Fig.
- In the susceptible.
- Aphids induced expression of genes in defense pathways in the 6-wk resistant genotype as well.
- However, the magnitude of these induc- tions was lower than in the younger sorghum (Fig.
- Bio- synthesis of secondary metabolites and phenylpropanoid pathway were also enriched in the 6-wk susceptible sor- ghum (Fig.
- 3 Enriched Gene Ontology (GO) terms in the two sorghum genotypes in response to aphid herbivory.
- [53] reported that synthesis of linoleic acid in response to thrips, Odontothrips loti Hali- day (Thysanoptera: Thripidae) increased only in the resist- ant alfalfa cultivar and not in the susceptible cultivar..
- For example, gene Sobic.001G393 500 from the CAM gene family showed higher expression in response to aphid herbivory regardless of genotype and age, while another gene from this family, Sobic.008G159100 was up-regulated in the 6-wk sorghum (Additional files 2, 3, 4 and 5).
- Further, the majority of mitogen-activated pro- tein kinases (MAPKs) genes including LPR receptor-like, and serine/threonine-protein kinase were up-regulated in response to aphid herbivory in the resistant genotype re- gardless of age.
- Signaling-related genes: Plant hormone signal transduction In hormone signal transduction pathways, the majority of DEGs (25 genes) were up-regulated in the 2-wk resist- ant genotype in response to aphid herbivory (Fig.
- In the.
- susceptible genotype, three NPR1 genes were up- regulated only in the 2-wk plants exposed to the aphids (Additional file 3).
- Several DEGs in the JA signaling pathway that regulates direct and indirect plant responses against herbivores [71–73] were also induced in our experiment.
- These genes had more than 2-fold change in response to aphid herbivory in the 2- and 6-wk resistant and suscep- tible genotype (Fig.
- We noted the greatest expression of NPR1 genes along with up-regulation of JAZ genes that act as negative regulator of the JA-signaling pathway in both 2- and 6-wk resistant and in the 2-wk susceptible genotype.
- On the other hand, JAZ genes were upregulated while NPR1 gene expression was unaffected in the 6-wk susceptible geno- type, which had the weakest overall transcriptional re- sponses to aphid herbivory.
- Abscisic acid is a plant hormone involved primarily in the perception of abiotic stresses which can be elicited by aphid herbivory [88, 89].
- Four genes in the ABA signaling cascade were up-regulated in the resistant 2-wk sorghum including Sobic.006G279100, Sobic.001 G350700 genes encoding SNF1-related protein kinase 2 (SnRK2, a positive regulator of ABA), and Sobic.003G19 8200, Sobic.001G424400 genes coding for type 2C pro- tein phosphates (PP2C, a negative regulator of ABA) (Additional file 2).
- up-regulated in the resistant 6-wk sorhgum, including bZIP23 transcription factor (Sobic.004G309600), which showed 3.2-fold induction in response to aphid herbiv- ory (Additional file 4).
- Sobic.009 G069700 and Sobic.002G055900 that act as a transcrip- tional repressor were up-regulated in the 2-wk resistant genotype in response to aphid herbivory, but only Sobic..
- 002G055900 gene was up-regulated in the susceptible 2-wk genotype (Additional files 2 and 3).
- change in allocation of resources from growth to defense and conferred greater resistance to the aphids in the younger plants.
- Induction of these genes in the 2-wk sorghum regardless of genotype but not in the 6-wk old sorghum suggests that the younger plants may be diverting their resources from primary to sec- ondary metabolism thus increasing their resistance to the herbivore.
- However, more research is needed to unequivocally link these transcriptional responses to decreased aphid performance in the younger and re- sistant sorghum..
- We found a number of significantly induced genes in cytokinine signaling pathway, several of which were induced in the absence of aphids and suppressed when aphids were feeding on the plants.
- Specif- ically, in the 2-wk aphid-free resistant sorghum, three posi- tive cytokinine regulator genes (Sobic.009G202900, Sobic.0 03G292600, and Sobic.004G330900) were up-regulated, in- dicating that plants were regulating their growth processes actively (Additional file 2).
- This outcome provides another example of the younger resistant sorghum switching from growth to defense in re- sponse to the aphids and highlights the lack of parallel re- sponses in the susceptible sorghum.
- Ethylene expression decreases with plant age, with a marked decline at the onset of reproduction asso- ciated with a significant reduction in the ability of plants to induce defenses [105].
- This was consistent with our results – we found ethylene gene expression only in the younger plants.
- Sobic.007G210700 and Sobic.009G050400, involved in the ethylene pathway were both up-regulated in the resistant 2-wk genotype in response to aphid and only Sobic.007G210700 was up-regulated in the susceptible 2-wk genotype (Additional files 2 and 3).
- Phenylalanine ammonia-lyase catalyzes the first step of the phenylpropanoid pathway, which is a key reaction in the control of lignin, flavonoid and salicylic acid biosynthesis [108].
- The up-regulation of a number of genes involved in glutathione metabolism pathway was mostly found in the resistant 2-wk sorghum in response to aphid herbivory (Additional file 2).
- [39], for example, reported that genes from cytochrome P450 family were up-regulated in the resistant genotype of soybean in response to aphid herbivory.
- Notably, we found these genes to be induced the strongest in the 2-wk resist- ant sorghum exposed to sugarcane aphids (Additional file 2), which may explain the superior host plant resist- ance observed in this genotype shortly after emergence..
- Specifically, we found the high- est number of differentially expressed TFs (52) in the resistant genotype and in the younger plants, and 18 of these TFs belonged to the WRKY family.
- In the 6-wk resistant genotype, a total of 42 TFs were up regulated in response to aphid herbivory, and 13 of these were WRKY TFs.
- A comparable number of WRKY genes were up- regulated in response to aphid herbivory in the 2- and 6-wk susceptible plants (Fig.
- It is noteworthy that four WRKY transcription factors were found to be expressed exclusively in the 2-wk resistant genotype exposed to the aphids, including WRKY1 (Sobic.001G095500), WRKY19 (Sobic.009G238200), WRKY28 (Sobic.003G199400) and WRKY72 (Sobic.005G117400) (Additional file 2).
- Sobic.003G337900, and Sobic.008G060300 were only expressed in response to aphid herbivory in 2-wk plants regardless of genotype, with higher expression in the resistant genotype.
- Real-time quantitative RT-PCR (RT-qPCR) of selected DEGs All genes selected for validation of RNA-seq data showed a similar expression trend (up-regulation or down-regulation) in the RT-qPCR analysis.
- 0.001), and aphid pop- ulations increased faster in the 6-wk old sorghum than in the 2-wk old sorghum (F .
- Aphid populations grew the slowest in the young resistant sorghum, and their numbers increased the most rapidly in the older susceptible sor- ghum.
- Further, these results indicate that the ro- bust transcriptional response to aphid herbivory in the younger resistant genotype is phenotypically reflected in the significantly diminished aphid performance on these plants..
- The host plant resistance reported previously for the resistant genotype and manifested in significantly lower aphid densities in the greenhouse and field experiments [32] was reflected at the transcriptome level in our ex- periment.
- sacchari herbivory, particularly in the resistant and.
- We detected a higher number of DEGs and expression of a higher number of defense related genes in the resistant genotype in re- sponse to aphid herbivory than in the susceptible geno- type.
- The oxidation reduction GO term was highly enriched in the resistant genotype com- pared to the susceptible genotype.
- We also found that pathways coding for secondary metabolites, plant hor- mone signal transduction, plant-pathogen interaction, and glutathione metabolism were over-expressed in the resistant genotype.
- Our results suggested a potential cross-talk between SA and JA in the resistant genotype such that up-regulation of SA pathway genes negatively affected JA pathways genes through higher expression of JAZ genes that act as a negative regulator of JA biosynthesis pathway genes.
- Similar differences were noted when gene ex- pression was compared between the 2-wk and 6-wk old sorghum – the number of DEGs, enriched GO terms, and significantly affected pathways were higher in the 2-wk old plants than in the 6-wk old sorghum.
- In conclusion, sugarcane aphid herbivory elicited the most robust transcriptional changes in the younger re- sistant genotype, and several pathways and specific genes provide insights into the mechanisms underlying host plant resistance to this invasive insect.
- Lack of parallel responses in the older sorghum plants has relevant implications for aphid-sorghum in- teractions, and suggests lower host plant resistance in sorghum colonized at the onset of the reproductive stage.
- Further, unique induction of four WRKY genes only in the 2-wk resistant genotype highlights the likely importance of these TFs in the superior resistance of.
- The colony was maintained in the greenhouse for one year prior to the experiment.
- Further, the COI sequencing confirmed that the aphids were of dif- ferent haplotype than previously described in sugarcane in the U.S.
- Six-wk old plants were exposed to aphids in the same manner, with the exception of tissue collection for RNA extraction – only leaves exposed to the aphids and not entire plants were collected.
- For functional annotation, GO analysis was performed using AgriGO gene ontology analysis tools [132] to determine overrep- resented GO categories in the up- and down-regulated DEGs, and significantly enriched GO terms were identi- fied.
- Experiments were conducted in the greenhouse follow- ing the protocol described above (Sorghum growth and M.
- Effects of sorghum variety, age, and the interaction between the two factors were in- cluded in the analyses, and means separation tests were performed were appropriate with a Bonferroni adjust- ment.
- We would like to thank Eva Huff for assistance in the greenhouse, Li Paetzold for help in the laboratory, and Kristyne A.
- Varela for assistance in the sample preparation.
- The funders had no role in the design of the study, collection, analysis, interpretation of the data, or in writing the manuscript..
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