Salivary Proteins

For protein identification of the mock community samples, a database was created using all protein sequences from the reference genomes of the organisms used in the mock communities (Supplementary Table 8). The cRAP protein sequence database (http://www.thegpm.org/crap/) containing protein sequences of common laboratory contaminants was appended to the database. The final database contained 123,100 protein sequences and is available from the PRIDE repository (PXD006118). For protein identification of the soda lake mats we used the database described above. For protein identification of the human saliva metaproteomes we used the same public databases as described in Grassl et al.^{9 (link)} as a starting point. Namely the protein sequences from the human oral microbiome database⁵³ and the human reference protein sequences from Uniprot (UP000005640). CD-HIT was used to remove redundant sequences from the database using an identity threshold of 95%^{49 (link)}. The saliva metaproteome database contained 914,388 protein sequences and is available from the PRIDE repository (PXD006366). For peptide identification and protein inference the MS/MS spectra were searched against the databases using the Sequest HT node in Proteome Discoverer version 2.0.0.802 (Thermo Fisher Scientific) or the MaxQuant software version 1.5.5.1^{15 (link)}.

A lectin microarray was produced by using 37 lectins (purchased from Vector Laboratories, Sigma-Aldrich and Calbiochem) with different binding preferences covering N- and O-linked glycans. The lectins were dissolved to a concentration of 1 mg/mL in the manufacturer's recommended buffer containing 1 mmol/L appropriate monosaccharide and spotted onto the homemade epoxysilane-coated slides, according to the protoco1, with Stealth micro spotting pins (SMP-10B) (TeleChem, USA) by a Capital smart microarrayer (CapitalBio, Beijing)31 (link)32 (link). Each lectin was spotted in triplicate per block, with triplicate blocks on one slide (Supplementary Figure S2). The slides were incubated in a humidity-controlled incubator at 50% humidity overnight and then put into a vacuum dryer for 3 h at 37°C to allow lectin immobilization. After incubation, the slides were blocked with blocking buffer containing 2% (w/v) BSA in 1 × PBS (0.01 mol/L phosphate buffer containing 0.15 mol/L NaCl, pH7.4) for 1 h, then rinsed twice with 1 × PBST (0.2% Tween 20 in 0.01 mol/L phosphate buffer containing 0.15 mol/L NaCl, pH 7.4), and followed by a final rinse in 1 × PBS. Afterwards, 4 μg of Cy3-labeled salivary protein was diluted in 0.5 mL of incubation buffer containing 2% (w/v) BSA, 500 mmol/L glycine and 0.1% Tween-20 in 1 × PBS and was applied to the blocked lectin microarrays and an incubation was performed in the chamber at 37°C for 3 h in a rotisserie oven set at 4 rpm. Each slide was washed twice with 1 × PBST for 5 min, washed once with 1 × PBS for 5 min, and then dried by centrifugation at 600 rpm for 5 min. The microarrays were scanned with a 70% photomultiplier tube and 100% laser power settings using a Genepix 4000B confocal scanner (Axon Instruments, USA). The acquired images were analyzed at 532 nm for Cy3 detection by Genepix 3.0 software (Axon Instruments, Inc. USA). The average background was subtracted, and the values less than average background ±2 standard deviations (SD) were removed from each data point. The median of the effective data points of each lectin was globally normalized to the sum of medians of all effective data points for each lectin in one block. Each sample was observed consistently by three repeated slides, and the normalized medians of each lectin from 9 repeated blocks were averaged and its SD was calculated. The normalized data of each clinical group were compared with the healthy groups based upon the fold change according to the following criteria: fold change >1.5 or <0.67 in the pairs indicated up-regulation or down-regulation, respectively, of a certain glycan. Differences between the arbitrary two data sets or multiple data sets were tested by Student's t-test or one-way ANOVA to each lectin signal using SPSS statistics 19 software.

Helper T- cell response is the major part of cell-mediated immunity and helps in pathogen clearance by the help of various cytokines and immune cells^{42 (link),43 (link)}. They have the ability to induce both CTL and humoral immune response by the secretion of lymphokines like IL-2, IL-4, IL6, Granulocyte-macrophage colony-stimulating factor (GM-CSF), and IFNγ. In view of that, we can say that HTL epitopes mainly of Th1 type are most likely going to be a crucial part of the prophylactic and immunotherapeutic vaccine. Therefore, IEDB MHC-II epitope prediction module was used to predict the HTL epitopes for all 14 Anopheles salivary protein sequences^{44 (link)}. The available parameters were kept default except for allele selection where the nominated alleles were H2-IAb, H2-IAd, and H2-IEd. Output epitopes were ranked based on their percentile rank score where lower percentile rank representing that greater will be the binding affinity for HTL receptor. Secondly, to prove our work that the predicted HTL epitopes will have ability to activate Th1 type immune response followed by the IFN-γ production, top 14 predicted HTL epitopes were subjected to the IFN epitope server using predict option. All 14 epitopes were submitted in the FASTA format followed by the approach selection and model of prediction. Motif and SVM hybrid was selected as the approach and IFN-gamma versus other cytokine as model of prediction.

Highly sensitive assay kits (R&D Systems) were used to determine protein concentrations in saliva samples. The tests were performed according to the manufacturer’s recommended protocols. The microtiter plate provided in the kits was pre-coated with a monoclonal antibody specific to the analysed protein. Standards and samples were added to the appropriate microtiter plate wells. Following incubation at room temperature, an enzyme-linked polyclonal antibody was added. Then, the microplate wells were aspirated and washed four times. Next, a substrate solution was added to each well. The enzyme–substrate reaction was terminated by addition of a stop solution and the colour change was measured spectrophotometrically at 450 ± 2 nm. The antigen concentration in the samples was determined by comparing the O.D. to the standard curve.

Salivary sheath samples and watery saliva samples were collected from 900 to 1000 nymphs as previously described^{14 (link),56 (link)}. Briefly, the 5th instar L. striatellus nymphs were transferred from the rice seedlings into a plastic Petri plate. Approximately 300 μl diets with 2.5% sucrose were added between two layers of stretched Parafilm, and the insects were allowed to feed for 24 h. Ten devices were used for saliva collection, with each device containing 90–100 L. striatellus. For the preparation of watery saliva samples, the liquid was collected from the space between two layers of Parafilm. To prepare salivary sheath samples, the upper surface of Parafilm with salivary sheath firmly attached was carefully detached, and washed in PBS thrice. As salivary sheath was difficult to dissolve, a lysis buffer of 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (#20102ES03, Yeasen), 2% SDS (#A600485, Sango Biotechnology) and 2% DTT (#A100281, Sango Biotechnology) was adopted for obtaining the solubilized salivary sheath proteins under gentle shaking on an orbital shaker at room temperature for 1 h according to previous description^{14 (link),56 (link)}. With this method, the majority of salivary sheath, although not all, can be dissolved^{56 (link)}. Since it was difficult to quantify the protein concentration in saliva solution, the salivary sheath samples and watery saliva samples was pooled to 50 μl using 3-kDa molecular-weight cutoff Amicon Ultra-4 Centrifugal Filter Device (Millipore, MA, USA), respectively.
The rice apoplast was collected with Buffer A (consisting of 0.1 mol/L Tris-HCl, 0.2 mol/L KCl, 1 mmol/L PMSF, pH 7.6) as previously described⁵⁷ . Briefly, 5.0 g rice plants were vacuum infiltrated with Buffer A for 15 min. Then, the remaining liquid on the surface was dried with the absorbent paper, placed inside the 1-ml tips and centrifuged in the 50-ml conical tubes at 1000 × g for 20 min. The apoplastic solution was concentrated using 3-kDa molecular-weight cutoff Amicon Ultra-4 Centrifugal Filter Device.
For the preparation of insect and plant samples, the insects/plants were collected at indicated time points and homogenized in the RIPA Lysis Buffer (#89900, ThermoFisher Scientific). To detect the secretion of LsSP1 into rice plants, approximately one hundred 5th instar nymphs were confined in the 2-cm stem and allowed to feed for 24 h. The outer rice sheath was collected for western-blotting assay.