Insect/pests are continuously evolving with respect to the repertoire of effectors they harbour. Effector proteins offer a selective advantage to polyphagous pests that helps to increase the range of host crops they infest. Their ability to suppress plant immunity by targeting key immune-responsive proteins make effector proteins an ideal candidate for pest management. Nevertheless, most of the studies on effectors, so far, have largely been focussed on sucking insects, while identification and characterisation of effector proteins of chewing insects is still in its infancy.
Spodoptera frugiperda, a chewing insect, is a destructive polyphagous pest of economically important crop plants. Since majority of the effector proteins are known to be secretory in nature, in the present study, we utilised the in-silico secretome prediction pipeline to identify a pool of S. frugiperda protein candidates bearing characteristics of secretory proteins. The predicted secretome will be crucial for the search of effector candidates of S. frugiperda. Out of 21,779 proteins analysed, 821 proteins of S. frugiperda were predicted to be secretory in nature, representing the secretome database of S. frugiperda (Fig. 1 and supplementary table- 2). Based on their annotated functions, the proteins of predicted secretome were divided into five groups i.e., i) proteases and protease inhibitors, ii) transcription factors and activators, iii) salivary secreted peptides and toxins, iv) receptors and signalling molecules and v) miscellaneous proteins (Fig. 2b). Tissue-specific expression analysis of 54 probable effector candidates (out of 821 proteins of predicted secretome of S. frugiperda) resulted in the identification of 13 candidates whose expression was limited to gut or salivary gland or both the tissues of S. frugiperda (Fig. 3a-iii,x,xi,xii ,3b-ii,v,vi,ix ,3c-ii,iv,v,vi,3e-ii).
Our strategy of using transcriptome database for generating S. frugiperda secretome by subjecting them to in silico prediction pipelines is in alignment with similar approaches used previously in diverse life forms. For instance, SignalP and TargetP bioinformatic tools were used to predict 884 small secretory proteins from in planta transcriptome of ascomycete Colletotrichum falactum expressed during the host-pathogen interaction (Prasanth et al. 2019). Similarly, a whole-body transcriptome mining approach in Russian wheat aphid, Diuraphis noxia led to the identification of 725 transcripts encoding putative secretary proteins including putative effector candidates of D. noxia (Nicolis et al. 2022). A dual transcriptomic-proteomic approach to generate a catalogue of candidate effector proteins from the salivary gland of pea aphid, Acyrthosiphon pisum led to the identification of > 300 proteins harbouring secretary signal (Carolan et al., 2011).
Furthermore, the functional groups identified in our analysis bear strong correlation with previous studies attempting to identify effector candidates from different organisms. For example, zymogens of serine proteases (one of the groups predicted in our screening) play an important role in activating effectors (Ross et al. 2003). A genome- and transcriptome-wide analysis of the brown plant hopper, Nilaparvata lugens, led to the identification of 90 putative serine proteases and serine protease homologs, which are predicted to be involved in defence responses besides other physiological functions (Bao et al. 2013). Another line of support comes from plant-fungus/insect interaction where serine proteases acts as effectors to counter plant chitinases secreted to degrade fungal and insect chitin (Karimi Jashni et al. 2015). Coherently, serine protease inhibitors present in the hemolymph of arthropods are predicted to protect them from infection by pathogens or parasites (Kanost 1999). Similarly, serpins (largest known member of Serine protease inhibitors) of parasitoid wasps and blood-feeding ticks and mosquitoes are involved in host pathogen interaction (Jiang and Kanost 2000; Meekins et al. 2018) Identification of ‘proteases and protease inhibitors’ in our analysis (Fig. 2b, 3a and supplementary table 2) is strongly supported by a recent in silico study performed for the identification of effectors in S. litura (Prajapati et al. 2020).
Transcription factors working as effector proteins in insects (Guo et al. 2022; Wang et al. 2022) bacteria (Moore et al. 2014) and fungus (Prasanth et al. 2019) is concurrent with the identification of several families of transcription factor, such as, GATA zinc-finger domain-containing protein 14-like, FLYWCH zinc-finger domain-containing protein, SPT20 homolog isoform X1 having "Chitin binding Peritrophin-A domain, myb-like protein Q and various REPAT proteins in our analysis (Fig. 3b and Supplementary table 2). RING finger domain (a protein structural domain of zinc finger type)-containing secretary proteins function as effectors in grapevine galling insect, Daktulosphaira vitifoliae (Zhao et al. 2019). Chitin binding ‘peritrophin-A’ domain protein lines the gut of most of the insects and protects them against microorganisms besides helping them in the process of digestion (Tellam et al. 1999). This protein is also predominantly found in ovaries of shrimps and is expected to function as effector protein as it contains signal sequences, and protect the eggs after spawning (Khayat et al. 2001). Interestingly, an independent analysis predicted chitin binding ‘peritrophin–A’ domain containing peptide in Tribolium castaneum using SignalP tool (Jasrapuria et al. 2010) further supporting its secretory nature. REPAT proteins (an immune-associated gene family) (Hrithik et al. 2021) picked up in our analysis (Fig. 3b and Supplementary table 2) have been shown to act as transcriptional co-activators in response to pathogens in S. exigua (Hernández-Rodríguez et al. 2009; Navarro-Cerrillo et al. 2013).
Another group predicted in our analysis is ‘salivary secreted peptides and toxins’ (Fig. 2b, 3c and Supplementary table 2). This is concurrent with the recently discovered Has1 (highly accumulated secretary protein 1) (Chen et al. 2023) and Harp1 (Helicoverpa armigera R like protein1) (Chen et al. 2019) that function as effector proteins and belong to salivary secreted peptides family. These peptides are related to venom-r-like proteins and is required by H. armigera for its successful infestation of plants. Moreover, secreted peptides and toxins have also been shown to provide significant fitness advantages to bacteria (Jeong et al. 2015) and fungi (Wang et al. 2014) against their rivals.
Receptors and signalling molecules identified in our analysis include macrophage mannose receptor 1-like, G-protein coupled receptor (GPCR) and GPCR-like receptor, Mth2-like receptor, arylphorin subunit alpha-like proteins isoform, Mindin proteins etc. (Supplementary table 2). Some of these proteins have been shown to be involved in biotic and abiotic defence responses, for e.g., Mth2-like isoform has been shown to be involved in insecticide resistance and toxicological responses in mosquitoes (Liu et al. 2021). Arylphorin subunit alpha-like protein was obtained in the secretome of Heliothis virescens as a defence response of Cry1Ac-intoxication (Castagnola et al. 2017). Further, Mindin, a member of the mindin-F-spondin family of secreted extracellular matrix protein, represents a unique pattern recognition molecule which initiates innate immune responses in mice (He et al. 2004; Lu et al. 2020).
The predicted secretome of S. frugiperda also harbours heat shock proteins (HSPs), chitin deacetylases, (Fig. 2b and Fig. 3e) Armet, apolipophorins and Waprin phi-like proteins (Supplementary table 2). Of these, HSPs, shown to provide cross protection and humoral immunity in insects (Dubovskiy et al. 2013; Richards et al. 2017; Wojda 2017), have been predicted in the secretome of Anisakis simplex (Kochanowski et al. 2022) and Trypanosoma species (Watanabe Costa et al. 2020) in previous in silico studies. Chitin deacetylases were recently predicted in the salivary secretome (generated using salivary transcriptome) of black-faced leafhopper, Graminella nigrifrons (Rajarapu et al. 2020). Authors hypothesize the role of chitin deacetylases in breaking down plant cell walls and in the reorganization of the salivary duct cuticle from one meal to another suggesting its possible function as effector protein. The possible effector function of chitin deacetylases is further supported by its identification in a study comparing the secretome of H. armigera fed on Arabidopsis or artificial diet (Chen et al. 2019). Armet, an effector protein, was identified in the salivary secretion of pea aphid, Acyrthosiphon pisum where it functions to induce transcriptional changes in pathogen-responsive genes of Nicotiana benthamiana (Wang et al. 2015). Further, Armet protein in the saliva of A. pisum was increased upon its feeding on natural diet (Vicia faba) as compared to that when fed on artificial diet. An independent study showcasing increased resistance in N. benthamiana against Pseudomonas syringae infection upon overexpression of aphid Armet further justifies the effector function of Armet (Cui et al. 2019). Apolipophorins have been found to be present in the secretome of N. lugens (Liu 2016), Nephotettix cincticeps (Hattori et al. 2015), D. noxia (Nicholson et al. 2015) and in the salivary secretions of aphids (Vandermoten et al. 2014). Apolipophorins activate immune responses in insects (Whitten et al. 2004) and are known to provide protection against Cry3Ba, Cry1Ca proteins of Bacillus thuringiensis (Contreras et al. 2013).
The tissue-specific expression analysis highlighting salivary gland- or gut-exclusive expression (Fig. 3a-iii,x,xi,xii ,3b-ii,v,vi,ix ,3c-ii,iv,v,vi,3e-ii) of some of the candidate genes of S. frugiperda secretome is suggestive of the corresponding encoded proteins being potential effectors of this insect. This is because salivary gland of insect serves as the reservoir of effector proteins (Naalden et al. 2021). Consistent with this, many previous studies have led to the discovery of effector proteins from the salivary gland of insects. Notwithstanding, the genes which amplified in all the tissues, or which did not amplify in gut or salivary gland, or both the tissues cannot be excluded from the list of potential effector proteins of S. frugiperda, as effector proteins have been reported even from non-salivary tissues, such as, eggs and frass of insects (Basu et al. 2018). Our hypothesis is further supported by the fact that many insect effectors are overexpressed/secreted in response to specific diet (diet/plant-specific effector proteins).
The study presented here is the first report of the prediction of the secretome of S. frugiperda. The methodology used in this study has opened up new avenues for the prediction of secretome from other economically important insect/pests where extensive proteome database is not available. Besides, S. frugiperda secretome prepared in this study will work as a catalogue for the identification of effectors of S. frugiperda. Future studies aiming to characterise the proteins of S. frugiperda secretome will shed more light on their secretory and effector potentials thereby paving ways for improved insect/pest management strategies.