Transcriptome analysis of cassava (Manihot esculenta Crantz) in response to mealybug (Phenacoccus manihoti) infestation

Abstract

Cassava mealybug (Phenacoccus manihoti) is one of the most damaging pests of cassava (Manihot esculenta Crantz) globally. While biological control of mealybugs through the use of natural predators has been successfully practiced, breeding for resistant cultivars remains an important means of control. Understanding plant responses to insect herbivory, by determining and identifying differentially expressed genes (DEGs), is a vital step towards the understanding of molecular mechanisms of defence responses in plants and the development of resistant cultivars by gene editing. Mealybug isolates were collected from different cassava growing regions of South Africa and a detailed morphological and molecular characterization of the pests was undertaken. Morphological and molecular analysis confirmed the mealybug identity as Phenacoccus manihoti (Matile-Ferrero). The positively identified mealybug isolates were used to artificially infest three cassava genotypes with contrasting response to mealybug. The response of cassava genotypes AR23.1 (which has resistance to multiple diseases and to green mites), P40/1 (which has no known resistances) and 98/0581 (which has multiple pest resistances and resistance to cassava mosaic disease) was further investigated by analysing their transcriptome after mealybug infestation at 24 and 72 hours in comparison with non-infested mock tissues. A total of 301, 206 and 269 transcripts from AR23.1, P40/1 and 98/0581 were differentially expressed (log2 fold and P ≤ 0.05) per time point following mealybug infestation. In two genotypes (AR23.1 and 98/0581), mealybug infestation resulted in a higher number of down-regulated than up-regulated genes between mealybug-infested and mock-infested tissues, while in P40/1 between infested leaves at 24 hpi and 72 hpi, more genes were up-regulated than were down-regulated. There was an increase in the number of DEGs from 24 hpi to 72 hpi suggesting an early induction of defence responses during the mealybug infestation. Gene expression was further compared between AR23.1, P40/1 and 98/0581 to determine whether there were any genotype-specific expression patterns. Within the Gene Ontology (GO) functional classification, DEGs in the class Secondary Metabolic Processes were significantly enriched in AR23.1 in comparison with P40/1 and 98/0581. Additionally, DEGs in the class Regulation of Molecular Function were significantly enriched in P40/1, while DEGs in the classes Reproduction and Reproductive Processes, and Nutrient Reservoir, were enriched in the multi-pest resistant 98/0581 genotype compared to P40/1 and AR23.1. In the metabolic pathway analysis performed using the KEGG database, DEGs in the classes ABC Transporters and MAPK signalling pathways were present in AR23.1 and 98/0581; and not in P40/1. There were both up- and down-regulated genes that were mapped to the classes Plant-Pathogen Interactions, and Plant Hormone Signal Transduction pathways in genotypes AR23.1 and 98/0581; while in P40/1, there were only down-regulated genes classified under Plant-Pathogen Interactions pathway. The up-regulated DEGs associated with these pathways are suggested to be involved in defence in response to P. manihoti feeding since these stress-associated genes, including those related to transcription factors, phytohormones and secondary metabolism were significantly induced in the AR23.1 (resistant) and 98/0581 (tolerant) genotypes, and not in the P40/1 (susceptible) genotype. Stress-associated genes such as 2-oxogluterate / Fe (II)-dependent oxygenase superfamily protein, terpene synthase 21, heat shock family proteins and cytochrome P450 superfamily proteins were induced in AR23.1 and 98/0581 genotypes. The results revealed a significantly different response to mealybug infestation in the three genotypes studied, with genotypes AR23.1 and 98/0581 showing a higher proportion of differentially expressed transcripts compared with the susceptible genotype P40/1 during infestation with mealybugs. Putative candidate defence-related genes that were overexpressed in the AR23.1 genotype post-infestation will be useful in future functional studies towards the control of mealybugs. These results will further form the basis for more detailed future studies on the specific role of all the differentially expressed transcripts identified. The transcripts identified in this study will also contribute significantly to the cassava EST database, and can be used to improve the annotation of the cassava genome.