MAMP-triggered resistance induced by elicitor protein PeBA1 derived from Bacillus amyloliquefaciens NC6 in common bean (Phaseolus vulgaris L.) against green peach aphid (Myzus persicae Sulzer)

Bacterial microbe-associated molecular patterns (MAMPs) play an important role in innate plant immunity. This in vitro study evaluated the putative role of protein elicitor PeBA1 derived from Bacillus amyloliquefaciens NC6 strain in eliciting induced resistance type responses in common bean (Phaseolus vulgaris) plants against green peach aphid Myzus persicae. Nymphal developmental time of aphids was significantly prolonged and the fecundity was significantly reduced by different concentrations of PeBA1 elicitor (i.e. 40.51, 24.91 and 16.38 μg mL) applied at three different temperature regimes (i.e. 21, 27 and 30 °C). Moreover, foliar application of PeBA1 elicitor protein strongly up-regulated the expression levels of salicylic acid (SA) pathway-associated genes, while the expression levels of jasmonic acid (JA) pathwayassociated genes exhibited a moderate induction. Quantification by LC/MSMS revealed a linear increase of both SA and JA plant defense hormones along with the time of exposure. Our findings suggest that the bacterial elicitor protein PeBA1 could be used as an effective biological pest management tool against phloem-feeding insect pests such as green peach aphids M. persicae.


Introduction
In nature, plants are confronted by a wide range of pathogens and herbivore pests. In response to these biotic stresses, they have evolved different physiological and molecular defense mechanisms including hypersensitive response (HR), defense signaling pathways, reactive oxygen species (ROS) and reactive nitrogen AcademicPres Notulae Botanicae Horti Cluj-Napoca Agrobotanici species (RNS) synthesis (Delledonne et al., 2001;Garcia-Brugger et al., 2006). These plant defense pathways are regulated by transcriptional and metabolic changes through physiological reactions, and are often directly or indirectly activated by different signaling molecules such as nitric oxide (NO) and ROSs produced by the activation of plasma membrane proteins or protein phosphorylation (Garcia-Brugger et al., 2006).
Apart from plant-derived compounds, plant defense reactions are also elicited by a number of pathogenic microbe-or herbivore-derived molecular patterns (MAMPs or HAMPs) which ultimately induce systemic and local resistance against the pests (Durrant and Dong, 2004;Maffei et al., 2012). Various MAMP type elicitors have been extracted from different microorganisms such as fungi, bacteria and viruses and are usually glycoproteins, oligosaccharide, lipids and proteins (Boller and He, 2009;Ellis et al., 2009;Maffei et al., 2012). Most of these elicitor molecules are perceived by plants under attack as signal molecules to defend themselves through inducing resistance against attacking herbivore pests and/or pathogens via activation of different plant defense pathways (Bostock et al., 2001).
Plant hormones play a major role in triggering host plant defenses against different abiotic and biotic stresses such as cold, heat and drought stresses and herbivore infestation or pathogenic infection. For instance, salicylic acid (SA) and jasmonic acid (JA) are two most important signaling molecules that elicit and enhance various plant defense responses (Thaler et al., 2012). Commonly, SA has been found associated with resistance to biotrophic pathogens and herbivory by phloem-feeding insect pests, while JA is usually induced in plants stressed by necrotrophic pathogens and chewing insect pests (Glazebrook et al., 2005;Smith et al., 2009). Moreover, expression studies of classical JA-and JA-associated marker genes have shown the antagonistic biosynthesis and signaling of both these plant hormones (Mur et al., 2006;Loake and Grant, 2007).
Although, entomopathogenic fungi constitute an important biological control strategy against different insect pests (Vega et al., 2012), their ability to grow endophytically within different plant portions has suggested their new ecological functions (Quesada-Moraga et al., 2014;Nazir et al., 2019). For instance, these fungi may induce systemic resistance in plants against different biotic and abiotic stresses (Waller et al., 2005;Sánchez-Rodríguez et al., 2018), suggesting their new perspective to develop novel plant protection strategies (Sánchez-Rodríguez et al., 2016). Recently, a number of elicitor proteins have been isolated from different biotrophic and necrotrophic fungal species exhibiting induced resistance against herbivore pests and pathogens. For instance, the elicitor proteins PeBC1 and PeBA1 cloned respectively from a necrotrophic fungus Botrytis cinerea and bacterial strain Bacillus amyloliquefaciens NC6, induced resistance against diseases in Arabidopsis (Zhang et al., 2014) and in tobacco (Wang et al., 2016).
Keeping in view these novel ecological aspects of microbes, this in-vitro study was carried out to determine the biological activity of an elicitor protein PeBA1 derived from the gram-positive bacteria B. amyloliquefaciens NC6 strain against green peach aphids M. persicae. This elicitor protein consists of 285 amino acid residues and is capable of inducing systemic resistance features in tobacco plant including transcription of JA and SA related defense genes, cell death, HR necrosis and ROS synthesis (Wang et al., 2016). Moreover, as the abiotic factors such as temperature may influence the JA and SA signaling pathways (Zhu et al., 2010), bioassays were carried out at three different temperature regimes in order to assess the response of aphids to PeBA1 elicitor protein.

Insect and plant culture
In a growth chamber, a colony of green peach aphid M. persicae was maintained on common bean (Phaseolus vulgaris L.) plants in isolation cages. In order to ensure that aphids have been properly adapted to the chemistry of bean plants, they were maintained in a controlled chamber at 65 to 75% relative humidity and 21 ± 2 °C temperature on the bean plants for at least three months prior to the start of actual bioassays. Bean plants were grown in plastic pots with sterile commercial potting mix and were raised in a greenhouse under natural light with day and night temperature fluctuating between 21 to 33 °C (from June to September). Prior to experimentation, short-term feeding bioassays were conducted in order to observe the suitability of foliage from the control and elicitor-treated bean plants for aphid growth and development. Experimental bean plants were grown in 15 cm plastic pots filled with sterile commercial potting mix (manure and soil in 1:5 ratio). Ten days old plants at 3-leaf stage were used in bioassays.

Protein purification
The elicitor protein PeBA1 was extracted from the colony of bacterial strain Bacillus amyloliquefaciens NC6 cultured in 1 L of LB medium for 12 h at 16 °C at 200 rpm. The pellets were then collected, broken and re-suspended by sonication. Then, centrifugation of the solution was performed for 15 min at 12000 rpm followed by the collection and filtration of supernatant through a 0.22 µm filter paper. Final purification of elicitor protein was carried out with help of affinity chromatography using a His-trap HP column and A, B, C and D loading buffers. Buffer A (50 mM Tris-HCl, pH 8.0) was used to wash the column, while buffer B (50 mM Tris-HCl, 200 mM NaCl; pH 8.0) was used to stabilize the column. Buffer C (50 mM Tris-HCl, 200 mM NaCl and 20 mM imidazole; pH 8.0) and buffer D (50 mM Tris-HCl, 200 mM NaCl and 500 mM imidazole; pH 8.0) were used to elute the elicitor protein. Elicitor protein was then desalted by desalting tubes and by centrifugation at 4 °C at 5000 rpm. The molecular mass of purified recombinant elicitor protein was determined by 10% SDS PAGE gel and by using a protein marker.
Bioassays with PeBA1 elicitor protein against aphids Different concentrations of purified PeBA1 elicitor protein were bio-assayed against M. persicae aphids on bean plants. Treatments included three different concentrations of elicitor protein (i.e. 40.51, 24.90 and 16.38 µg mL -1 ) and the control in which plants were treated only with buffer A (50 mM Tris-HCl, pH 8.0). Protein concentrations were determined by Bradford assay using bovine serum albumin (BSA) as standard. Elicitor treatments were applied on bean plants at 3-leaf stage with a precise spray bottle until the bean plants were thoroughly covered and began to drain. Three milliliters of each elicitor solution were applied on each plant. Control plants were sprayed with buffer only. Plants were allowed to dry overnight and next day five freshly molted (0-6 h old) adult winged aphids per leaf were released on these plants. Nymphal development time for each instar and total number of offspring produced by these F1 aphid progeny were recorded by consecutive observations at 3 h intervals until the completion of bioassays (in approximately 3 weeks). Each treatment was replicated 10 times and the bioassay was repeated thrice at three different temperature regimes (i.e. at 21, 27 and 30 °C) independently.

Isolation of RNA and cDNA synthesis
For the molecular characterization of PeBA1 elicitor-induced resistance in plants against M. persicae aphids, bean plants were treated with the highest elicitor concentration (i.e. 40.51 µg mL -1 ), and were allowed to be infested by aphids as described in previous bioassay. Leaf samples were then collected from both treated and un-treated plants and RNA was extracted from these leaves using plant RNA kit Trans ER 301-01(All Style Gold Tech. Co. Ltd., Beijing, China) following manufacturer's protocol. Quantity and quality of the extracted RNA were measured with Nano Photometer (NP80-Touch, Implen Inc., NP West Burg, USA). Later on, this extracted RNA was reverse transcribed to cDNA using one step gDNA and cDNA kit Trans AT 341-01 (All Style Gold Tech. Co. Ltd., Beijing, China) following manufacturer's protocol.

RT-qPCR analysis
Using RT-qPCR, the relative expression of genes putatively involved in JA and SA plant defense pathways were determined in the elicitor-treated and buffer-treated bean plants. Marker genes involved in the JA pathway included phvul.001G000800g, phvul.001G001300g, phvul.001G017800g, phvul.002G175500g, phvul.002G06700g, phvul.003G011600g, phvul.003G111500g and phvul.003G096400g. Similarly, the SAassociated marker genes used in the study were phvul.006G048600g, phvul.008G272800g, phvul.008G057700g, phvul.009G022200g and phvul.011G176100g. Actin was used as the reference gene (internal control). Ten folds diluted cDNA was used as a template. Primer pairs used for the RT-qPCR amplifications of these genes are given in Table S1. The efficacy and specificity of all primer pairs were validated through qPCR and gel electrophoresis. Thermocycler ABI 7500 (Applied Biosystems, USA) was used for RT-qPCR amplifications. Each reaction mixture was 20 µL holding 2 µL of cDNA template, 0.5 µL of each 10 µmol L -1 forward and reverse primer, 10 µL of 2× SYBR ® Premix Ex Taq (Takara, Dalian, China) and 7 µL of ddH2O). PCR thermal program was constituted of preheating at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 30 s and annealing at 60 °C for 40 s and elongation at 72 °C for 60 s. There were three biological and three technical replicates for each sample.

LC-MS/MS analysis
Apart from the expression analysis of potential JA-and SA-associated genes, we quantified these plant hormones in the leaves of aphid infested elicitor treated and buffer-treated plants using LC-MS/MS chromatography. For this determination, leaves of bean plants were collected at different time intervals (i.e. 6, 12, 18 and 24 h) and were frozen dried using liquid nitrogen. Leaf samples were then placed into 2 mL microcentrifuge tubes and were ground in a bead-beater using 2 mm tungsten-carbide beads with 25 Hz/s for 4 min. About 20 mg of powdered tissue (equivalent to almost 200 mg fresh weight) was extracted with 400 µL of 10% methanol and 1% acetic acid. Extraction was repeated twice recovering about 90-95% of the sample. Both plant hormones were determined using QTRAP ® 5500 LC-MS/MS System (Applied Biosystems, Foster City, CA, USA) equipped with a 1290 Series HPLC System (Agilent Technologies, Foster City, CA, USA) and Turbo Ion Spray Electrospray Ionization (ESI) source. Quantifications were performed using multiple reactions monitoring (MRM) method in a negative ion detection mode (m/z 209.1/59.2 for JA and 137.0/65.1 for SA). The retention times for JA and SA were 5.02 and 4.47 min, respectively.

Statistical analysis
The experiments were repeated three times independently and the mean values of all parameters are presented in figures along with standard errors. Statistix Version 8.1 (Analytical Software, Tallahassee, FL, USA) was used for statistical analyses of data. Some data, for instance of fecundity of aphids, was subjected to square-root transformation prior to analysis. One-way analysis of variance was run to find out significant differences among treatment factors followed by Tukey's HSD test at 95% level of probability. The RT-qPCR expressions of genes were measured using comparative CT (2 -∆∆CT ) method. The fold change in the elicitortreated and buffer-treated (control) plant samples was compared by Student's t-test at α = 0.05.LC-MS/MS data acquisition and processing were carried out using AB Sciex (V 1.6.2) software and were analyzed by regression analysis.

Influence of PeBA1 elicitor on nymphal development time of aphids
Analysis of variance showed a significant impact of different concentrations of PeBA1 elicitor protein (F3, 468 = 12.71; p < 0.001), temperature regimes (F2, 468 = 274.27; p < 0.001) and of their interaction (F6, 468 = 5.61; p < 0.001) on the overall developmental time of M. persicae (Table S2). The effect of protein elicitor on nymphal development time of aphids was found different at different temperature regimes. The developmental time of each nymphal instar was prolonged along with the concentration of PeBA1 elicitor. Maximum nymphal developmental time was recorded as 4.8 days for 1 st instar to 6.4 days for 3 rd instar nymphs for high concentration (40.51 µg mL -1 ) at low temperature (21 °C). While minimum nymphal development time (1.8 days) was recorded for 1 st instar for low elicitor concentration (16.38 µg mL -1 ) at high temperature regime (30 °C). In buffer-treated (control) plants, the nymphal development time varied from maximum (4.2 days) for 3 rd instar at 21 °C to minimum (1.7 days) for 1 st instar at 30 °C ( Figure 1).

Effect of Elicitor PeBA1 on the fecundity of aphids
Results showed that different concentrations of PeBA1 elicitor (F3, 108 = 23.08; p ≤ 0.001) and temperature regimes (F2, 108 = 225.72; p ≤ 0.001) significantly influenced the fecundity of M. persicae adults (Table S3). Aphid individuals fed on the elicitor-treated plants produced significantly less number of offspring during their lifespan than those fed on the control (buffer-treated) plants. Moreover, minimum fecundity was observed at high temperature (30 °C), while the maximum fecundity was observed at low temperature (21 °C) ( Figure 2). For each temperature regime, different alphabets above bar tops indicate significant differences between treatments (one-way ANOVA; Tukey's HSD at α = 0.05) Effect of PeBA1 elicitor on expression of JA and SA pathway-associated genes Results revealed differential expression levels of different key genes associated with JA and SA pathways in elicitor-treated bean plants as compared to control ones. All SA-associated genes were highly up-regulated and were significantly different from the control samples for all observation times (Figure 3). Although relatively moderate but a similar trend of expression levels was observed for all JA-associated genes. Most of these genes were up-regulated at all observation times. However, one gene (phvul.002G175500g) initially showed no up-regulation till 6 h post aphid infestation and then was up-regulated from 12 h onward ( Figure  4). Moreover, maximum gene expression levels for both types of key genes were recorded at 18 and 24 h of the elicitor application and aphid infestation.

Quantification of JA and SA hormones
Results of LC-MS/MS quantification of plant defense hormones have shown that levels of both JA and SA were gradually and significantly increased along with time. Maximum hormone amount was recorded after 24 h of elicitor treatment, while minimum was recorded at 6 h. In control plants, no significant amount of JA was recorded, while a considerable quantity (2.30 µg g -1 ) of SA was observed in control plants ( Figure 5). A positive correlation was found between these two plant hormones and time of exposure to protein elicitor PeBA1 (R 2 = 0.98 for SA and 0.82 for JA).

Discussion
Elicitors play a vital role in signaling defense mechanisms in plants under attack and are considered as a novel biological pest management approach. Pathogenic fungi, either biotrophic or neurotropic, constitute a major source for microbes-derived elicitors such as PAMPs or MAMPs (Zhang et al., 2014;Wang et al., 2016). This in-vitro study elucidated the activity of an elicitor protein PeBA1 derived from B. amyloliquefaciens NC6 strain for the control of aphid M. persicae. The results of our bioassays have demonstrated that aphid population developed significantly slower on elicitor-treated plants than on the buffer-treated control plants. Our results are consistent with the previous studies demonstrating the negative impact of exogenous applications of different plant defense inducing chemicals including JA, benzothiadiazole (BTH) and methyl jasmonate (MJ) on the population growth and fitness parameters of different aphid species (Cooper and Goggin, 2005;Boughton et al., 2006). Similarly, our results corroborate the findings of Mallinger et al. (2011) who documented that methyl salicylate elicitor reduced the population of soybean aphid Aphis glycines up to 40%. Similarly, some studies have shown that the activity of herbivore insect pests was significantly reduced after the application of plant defense inducing molecules such as MJ and BTH and other plant defense proteins such as proteinase inhibitors in tomato crop (Bostock et al., 2001;Bale et al., 2002). Exogenous application of elicitor molecules usually induce systemic or local resistance in plants against herbivore insect pests most probably by activating the plant SA and JA associated pathways resulting in the induction of different plant defense proteins such as peroxidase (POD), polyphenol oxidase (PPO), PR proteins, lipoxygenase and protein inhibitors etc.is well known (Bostock et al., 2001;Maffei et al., 2012).
Although PeBA1 elicitor application caused reduced nymphal developmental time and fecundity of aphids, the impact is statistically more significant and pronounced at low temperature (21 °C). Maximum nymphal development time was observed at lower temperature (21 °C) confirming the fact that with the increase of one degree in temperature, insect life cycle would become shorter (Bale et al., 2002). Similarly, the application of PeBA1 elicitor reduced the aphid fecundity. Aphids reproduced much lower on the elicitor treated plants as compared to buffer treated control plants. These results are in line with the previous studies evidencing a lower mean lifetime fecundity of aphids induced by the exogenous application of SA (Mahmoud and Mahfouz, 2015) and MJ (Boughton et al., 2006).
Our study revealed the potential of PeBA1 elicitor on the suppression of population performance and growth parameters of sap-feeding herbivores. However, as different chemical elicitors such as JA and MJ may induce the production of various proteinase inhibitors in plants as described in tomato plants (Farmer et al., 1992), more studies are needed to better comprehend the underlying mechanisms induced in bean plants by the PeBA1 elicitor protein which actually influenced the nymphal developmental time and fecundity of aphid individuals.
Moreover, SA and JA hormones play an essential role in signaling plant defense pathways and in the regulation of defense associated genes (Moran and Thompson, 2001;Thaler et al., 2012). These molecules enhance plant defense responses including resistance against insect herbivory and pathogenic attacks (Ali and Agrawal, 2012). In this study, we found that elicitor PeBA1 produced a significant and strong up-regulation of all key genes potentially associated with SA pathway, while a moderate up-regulation was observed in case of JA pathway associated genes. Our findings are in agreement with the fact that phloem-feeding insect pests such as aphids trigger more strongly the expression of SA pathway associated genes than those related to JA pathway (Smith et al., 2009;Ali and Agrawal, 2012;Coppola et al., 2013). This is further supported by the results of liquid chromatography-mass spectrometry which showed some quantity of SA contents in aphid infested control plants as compared to the negligible JA contents. This corroborates the fact that herbivory by sapfeeding pests such as aphids usually triggers more strongly the key genes associated with SA plant defense pathway rather than those involved in the JA pathway (Boughton et al., 2006;Coppola et al., 2013). Moreover, our results are in line with our recent study  which demonstrated a significant sub-lethal effect of PeBC1 protein elicitor, derived from the fungus Botrytis cinerea, on M. persicae concomitantly with a pronounced up-regulation of the expression level of genes related to SA and JA pathway.

Conclusions
In this study, we postulated that the application of PeBA1 elicitor would prolong the nymphal development time and reduce the fecundity of M. persicae on common bean plants. Bioassays performed with different concentrations of PeBA1 applied at three different temperature regimes revealed that aphids developed and reproduced slower on elicitor-treated plants than on control plants. Furthermore, functional characterization of key genes potentially involved in JA and SA plant defense pathways indicated that elicitor PeBA1 induced a significant and differential expression of genes in bean plants concomitantly with an enhanced production of both hormones. Based on overall results of the study, it is concluded that MAMPs such as PeBA1 could be effectively used as novel biological pest control tool against aphids and other phloemfeeding insect pests.