Plants resist herbivorous insects through a combination of constitutive or induced defenses that are generally manifested through poor feeding, abnormal development, low fecundity or even mortality. Various molecular and biochemical approaches can be used to determine the role of constitutive or induced plant defense responses against herbivory [1
]. These approaches are equally useful to reveal complex plant-insect interactions that may assist in identification of candidate genes involved in plant defense response [4
Rice is susceptible to a number of insect pests that affect its yield and quality; consequently, several modern rice varieties have so far selectively been developed with resistance to insect pests [6
]. Resistant varieties differ considerably in their responses to guard against pests particularly due to the presence of resistant (R) genes. For instance, rice varieties may be bred with R genes for resistance to stem borers, planthoppers or a combination of genes for resistance against multiple pests. Nevertheless, the induction of plant defense mechanisms that includes the production of nutritional and defensive proteins, phenolic compounds or protease-inhibitors and so will strongly contribute towards protecting the plants against insect damage [4
]. Although the presence of R genes potentiates rice defense mechanisms against herbivores, the role of other non-R gene like mechanisms and their mutual interaction with R genes during herbivory cannot be excluded [6
]. Broadly speaking, the overall resistance to insect infestation will be a cumulative response of different cellular processes in the plant, including input of R and non-R genes that may be interacting particularly during stress to help the plant express their defense response. Elucidating the complex phenomena of rice defense is will be important to plan rice resistance strategies for existing and emerging pests.
The brown planthopper (BPH), Nilaparvata lugens
Stål (Hemiptera: Delphacidae), is a secondary pest of rice and causes significant economic loss to susceptible rice cultivars [10
]. Continuous feeding by BPH populations for several days on rice in the field may lead to hopperburn, a condition resulting from wilting of tillers [9
]. Growing resistant varieties of rice is considered the most effective and environment friendly way to control the BPH. So far, more than 20 rice genes and quantitative trait loci (QTLs) have been identified and introduced to various cultivars through breeding in order to confer BPH resistance [11
]. Rice resistance through the introduction of QTLs has been shown to be effective against BPH [13
]. However, due to the genetic complexity between resistant rice cultivars, it has been difficult to explain the function QTLs play in the resistance mechanisms against BPH that further hinders the performance of resistance cultivars in different environments. Expression analysis of global genes and proteins is one strategy to understand molecular responses of rice plants during BPH stress to elucidate how different genes and proteins involve and interact during defense activities and help their selection for use in breeding rice resistance against BPH.
Rice defense against BPH has been well documented and the factors involved in rice resistance against BPH are usually associated with the differential regulation of genes and proteins during infestation [7
]. Many studies revealed physiological and metabolic changes in rice plants during BPH feeding [4
]. Such alterations in rice plant with BPH infestation also accompany transcriptional activation or repression of plant genes and reorganization of the gene expression profile during stress [7
]. It seems that not only the genes associated with cell defense are induced by BPH, genes that are involved in plant metabolism are also altered possibly through reallocation of necessary metabolites required for growth, reproduction, and storage towards defense activities instead [11
]. In this process, the genes associated with abiotic stress, pathogen stress and signaling pathways are reduced, whereas photosynthesis and defense related genes are increased [7
]. Extensive expression analysis of genes and proteins has facilitated the identification of several distinct genes affected by BPH feeding in rice that helped to differentiate susceptible vs.
resistant rice cultivars [9
]. For example, 160 unique genes were identified that responded to BPH infestation [15
]. Similarly, proteomics approach differentiated a susceptible line from a resistant line carrying a resistance gene BPH15 and identified additional eight genes differentially expressed in rice with BPH infestation [9
]. Advances in these tools and the ability to differentiate plant reaction to BPH stress suggests for a significant role expression analysis can play in developing rice resistance to BPH.
Mutational approach can play significant role in identifying proteins involved in rice response under specific physiological conditions such as abiotic and biotic stress [18
]. A comparative proteome analysis involving wild type rice and the mutants revealed contrasting differences in proteins induced in contrasting genotypes [19
]. Rice blast lesion mimic mutant (blm
) was differentiated from wild type plants based on pathogenesis-related class 5 and 10 proteins including a novel OsPR10d protein specific to the mutants’ response. This study also reported increase in phytoalexins and oxidative stress related marker proteins in blm
]. In another study, more than 150 protein spots were identified as differentially regulated between normal leaves of wild type and spotted leaves of the spl6 rice mutant, indicating the potential of proteomics to elucidate molecular response of rice [21
]. Proteomics of rice mutants, will certainly help to elucidate different proteins potentially involved in rice interaction with BPH and explain rice defense strategies against biotic stress [22
] This approach could be useful to explore QTL dependent resistance in rice cultivars such as IR64 and its mutants. IR64 is a modern rice variety developed at International Rice Research Institute (IRRI) that carries the major gene Bph1 and other minor genes located in a QTL responsible for resistance to BPH. The durable nature of BPH resistance in IR64 is thought to be due to synergy with minor genes, which contribute to a combined resistance through the mechanisms of antixenosis, antibiosis and tolerance [13
]. The mutants of this cultivar have been developed at IRRI [23
] and used for elucidating various physiological responses of rice.
The objective of the present study is to describe the proteomic responses of indica rice IR64 and two of its chemically generated mutants, one resistant and one susceptible to BPH infestation. Previous study with these IR64 mutants found no growth or yield penalty under normal field conditions [23
]. The contrasting phenotypes expressed by mutants that are essentially near-isogenic offer an opportunity to perform genetic analysis in response to BPH infestation and identify specific genes or proteins related to rice resistance. We performed a time-series analysis of gradual BPH stress on IR64 to identify BPH induced proteins. These proteins were further compared between wild type IR64 and the mutants to explain potential role of differentially altered proteins with BPH infestation.
Rice resistance to brown planthopper (BPH) is intricate involving genetically controlled defense mechanisms. Despite the existing knowledge of a large collection of rice genes, the molecular response involved in rice stress physiology particularly during interactions with BPH remained elusive. Mutants are valuable source of genetic diversity for gene discovery that could provide valuable information to explain plant defense mechanisms [18
]. We used mutants of the indica rice IR64 that differ in their response to BPH infestation to facilitate the understanding of rice resistance mechanisms to this economically important pest of rice. The time dependent differential change in the levels of BPH response proteins in rice helped to discriminate wild type with the mutants and revealed candidate proteins involved in plant resistance against BPH infestation.
Initially, the response of wild type IR64 was determined during BPH infestation, and proteins related to various functional categories were identified in BPH infested IR64; nevertheless photosynthesis, metabolism, and oxidative stress related proteins were predominantly altered (Table 2
). It has been reported that BPH infestation reduces photosynthetic activity in rice due to excessive loss of plant assimilates, decreased leaf area and wilting [11
]. Phloem feeding insects are generally known to alter the expression of genes required for photosynthesis [14
]. However, the role of housekeeping proteins such as those related to photosynthesis cannot be ruled out in defense against insects as housekeeping genes could shift their role towards defense metabolism to manage the increased energy demands during stress [29
]. For instance, photosynthesis-related genes altered during plant-insect interaction contributed towards defense needs while protecting the basic photosynthetic capacity [29
]. We also found a number of Rubisco large subunit fragments (RLSU) with BPH infestation. Similar observations have been reported with abiotic and biotic stresses in rice [31
]. Presence of several Rubisco large fragments (rbcl) with various experimental molecular weights and pI
s could also be due to oxidative stress induced fragmentation of the major Rubisco protein which is an abundant source of macronutrients such as nitrogen in senescing leaves [31
]. This supply of nitrogen during stress might serve as fuel for metabolic processes increased during BPH feeding stress.
We also observed changes in the levels of several antioxidant proteins that are known to scavenge excessive reactive oxygen species generated under stress [9
]. Some of these oxidative enzymes can be antinutritive to insects [37
]. Increased levels of oxidative enzyme activity might have adverse effect on the BPH performance thus helping to reduce damage. Similarly, generation of ROS can also act as stress signals to induce defense related genes during insect infestation [39
]. Few ascorbate peroxidase (APX) isoforms were found to be induced as early as 13 DAI (Figure 4
), indicating their primary importance during BPH infestation and implication in defense signaling. Moreover, we observed differential levels of APX related proteins in BPH infested IR64 as three of the APXs were increased whereas two were decreased during the infestation which is in agreement with previous studies on differentially induced ascorbate peroxidase isozymes during oxidative stress [40
Induction of proteins during stress is important in dealing with the stress-induced metabolic homeostasis through readjusting metabolic pathways and reallocation of plants’ resources for defense [41
]. During such response, proteins may be reduced or increased in activity as evidenced in this study. We observed 64 proteins induced with BPH infestation and 52 of these were identified (Table 2
), some of these might have role in higher energy demands during stress. This seems plausible as many of these proteins (Table 2
, Figures 4
), except for few non-rice proteins (#20, #38, #39, #39a, #40, #41, #68), are plant stress response proteins. These induced proteins could be by-products of stress metabolism or post translation modification but may also represent molecules needed in signal transduction or acclimation response of plants during stress [42
]. Fifteen proteins (Figure 5
) were observed only in BPH infested plants whereas these proteins were absent in controls. BPH induced proteins, some of which are still unknown, are potentially involved in rice defense during BPH stress. Induction of several other proteins (#23, #27 and #LD7) during BPH stress showed rice response similar to that observed in abiotic stress such as drought and salinity [25
]. Excessive loss of phloem sap and impaired water movement during BPH infestation leads to wilting like condition “hopperburn” which is the susceptible response of rice to BPH [9
]. Phloem feeding insects generally reduce foliar water potential in plants as a result of extensive feeding and results in the induction of transcripts associated with water stress [28
]. Any counter activity such as altered levels of abiotic stress related proteins that could to delay wilting may help to overcome BPH stress. Up-regulation of drought induced S-like RNase and salt stress induced proteins in BPH infested rice points the need for exploring these proteins in rice defense response to BPH stress.
Comparative analysis was performed to differentiate the proteome response of mutants from the IR64. Defensive response of mutants was demonstrated by differential pattern of proteins induced with BPH infestation. For example, abundance of stress induced glyoxalase I, known with plant defense activity [46
], was reduced in D1131 and IR64 but not to the same extent in D518 (Table 3
). A similar response was evident with GSH-dependent dehydro ascorbate reductase in D518. The protein EFTu1, similar to 45- kDa heat shock proteins with chaperone like activity [47
], was induced earlier (T2) and more intensely in D518 and IR64 (S Figure 1
) and its abundance was greater in D518 followed by IR64 and then D1131. EFTu1 has been reported as an important component of thermo-tolerance in maize and other environmental stresses [48
]. Another two proteins, S-like RNase and spot #27 were also more abundant in D518 in contrast to moderate levels of these proteins in IR64 and susceptible mutant D1131 (Table 3
). Higher levels of these proteins in D518 could be important in providing defense to D518 against increasing BPH stress. Similarly, abundance of certain proteins was highly reduced in D518 during BPH infestation whereas the decrease in protein levels was slow in IR64 and D1131 suggesting for higher metabolic shift or adjustment of metabolic pathways in the resistant mutant. On the contrary, some proteins were in greater quantities in D1131 than IR64 and D518 and may represent a susceptible response during BPH infestation (Figure S1
). Several antioxidant enzymes and their isoforms were affected with BPH stress. Differential modulation of antioxidant proteins in a resistant and susceptible rice line infested with BPH was previously reported [9
]. However, we could not differentiate IR64 resistance solely from its mutants based on antioxidant proteins such as APX as levels of these proteins were not different.
Differential induction of drought induced S-like RNase and salt stress induced proteins (spot #23 and #27) suggests for the relationship between rice resistance to BPH and abiotic stress that urges for exploring abiotic stress tolerant varieties against BPH and vice versa. S-like RNase genes constitute an important family of RNA-degrading enzymes that have been associated with phosphate starvation, ethylene responses, senescence and programmed cell death and defense against multiple stresses [25
]. Sticky digestive liquid from a carnivorous plant, Drosera adelae
, contained an abundant amount of S-like RNase which assists plants to obtain phosphates from trapped insects which help to defend them against microbes [52
]. Induced S-like RNase has shown to prevent the growth of fungal hypha in tobacco [53
]. It is likely that increased abundance of S-like RNase may play a role to protect the resistant cultivar D518 from BPH perhaps by inhibiting stylet or ovipositor movement in phloem sheath and reduced settling, feeding and egg laying has previously been observed [23
]. Further studies in this area will elucidate mechanisms that S-like RNase and other proteins might play in rice resistance to BPH. One option is to investigate the interaction of BPH induced rice proteins with in silico
structure analysis and molecular docking (to reveal complexity of rice response to BPH stress particularly for possible links to phosphate (Pi) starvation, plant-microbe interaction and drought. Further experiments with in silico
and transgenic approach will help to elucidate the precise role of BPH induced proteins in rice defense to BPH.
4. Experimental Section
4.1. Insect Culture and Plant Material
Brown planthopper (BPH), Nilaparvata lugens (Stal) populations were continuously maintained on the susceptible variety “Taichung Native 1” (TN1) at the International Rice Research Institute (IRRI), Los Baños, Philippines. The parent BPH population was collected from rice fields around IRRI, Laguna. Gravid females were used to get a synchronized hopper stage for infestation.
The Indica rice cultivar IR64 along with its two mutants, i.e.
, D518 (gain-of resistance) and D1131 (loss-of-resistance) generated through the chemical and radioactive mutagenesis of IR64 [23
] were used for this study. The mutant D518 shows enhanced resistance during BPH infestation whereas D1131 is susceptible. The mutants were used following six generations of selfing and after confirmed field evaluation showing absence of any deleterious effect of mutations. The field trials of these mutants revealed no agronomical differences from IR64 [23
] whereas analysis using IR64 specific molecular markers suggested that the mutants are essentially near-isogenic (unpublished data). The experimental plants were maintained under greenhouse conditions at 28 ± 2 °C with a photoperiod of 16 h day/8 h night cycle.
4.2. Plant Phenotype to BPH Infestation
Phenotypic response of IR64 to BPH infestation was determined using a modified seedbox screening technique under greenhouse conditions [13
]. This technique provides free choice to BPH nymphs to colonize the plants in the seedbox. Briefly, pre-germinated seeds were sown in seedboxes (45 cm × 35 cm ×15 cm) containing heat-sterilized soil in six equally spaced rows (two rows for each entry) and 15 seedlings per row. Each row (mutant or wild type) was randomized within a seedbox and replicated in three independent seedboxes. Ten-day-old seedlings were uniformly infested with 3–4 second-instar BPH nymphs per plant and allowed to settle on plants of their choice. Hopperburn symptoms were observed 34 days after infestation (DAI).
4.3. Proteomics Response after BPH Infestation
Since phenotypic response of IR64 differed with two mutants, a no-choice setup was planned to allow equal number of BPH stress to feed on these genotypes. Fifteen seeds of mutants or wild type plants were sown in individual nine inch circular pots using three technical and three biological replicates. The seedlings were maintained in the greenhouse and before infestation with three nymphs per plant 10 days after sowing, pots were randomized between entries and covered with mylar cage and infested. Control plants were not infested but were covered with mylar cage and arranged randomly. For protein extraction, the plants from three experimental and biological replicates were sampled at four time points after infestation. For the first sample (T1), plant tissue was harvested 2 DAI when the infested nymphs were still in 3rd–4th instar stage; the second sampling (T2) was done at 13 DAI when the majority of nymphs were at the adult stage; the third sampling (T3) was performed 28 DAI following the emergence of second generation nymphs; the fourth sampling (T4) was done when the susceptible mutant (D1131) started wilting (34 DAI). For protein analysis, a 10 cm sample above ground portion of leaf sheath was harvested and stored immediately in liquid nitrogen. For control, plants were harvested at same time points using non-infested plants.
4.4. Protein Analysis
Protein extraction. Total leaf sheath proteins were extracted in a precipitation solution (10% Trichloroacetic acid, 89.93% Acetone, 0.07% Dithiothreitol) using a modified method of Damerval et al.
]. The protein concentration was determined using a Protein-Assay-Kit (Bio-Rad) following the manufacturer’s instructions.
Protein separation and image analysis of 2D Gels. Gel electrophoresis was performed using non-linear (NL) 18-cm IPG strips with pH 4–7 and 3–10 (Amersham Pharmacia Biotech, Uppsala, Sweden). The IPG strips were rehydrated overnight in 350 μL of rehydration buffer and 100 μg of sample protein. The isoelectric focusing (IEF) of proteins was performed on a Multiphor II Electrophoresis unit (Amersham Biosciences) at 20 °C with constant 200 V for the first hour, 500 V for next 2 h and finally 16 h at 2950 V. Proteins from DTT/IAA equilibrated IEF strips were separated on 15% sodium dodecyl sulfate (SDS) polyacrylamide gels using a Protean-II Multi cell (Bio Rad: Hercules, CA, USA) at 4 °C.
The gels were stained with silver nitrate (Sigma Aldrich) for scanning or spot quantification analysis whereas coomassie blue stain (Sigma Aldrich) was used for protein identification with mass spectrometry using standard staining protocols. The gels were scanned with a GS-800 Calibrated Densitometer (Bio-Rad) at a resolution of 600. For spot detection, protein quantification and spot analysis, Melanie-3 image analysis software (GeneBio, Geneva, Switzerland) was used. Spot detection parameters were as follows: number of smooths, 5; Laplacian threshold, 5; partial threshold, 1; saturation, 90; peakness increase, 100; minimum perimeter, 10. The Melanie software automatically normalized the spot intensity (the relative volume) i.e., the volume divided by the total volume over the whole image (Melanie 3 user manual). The percent spot volume detected by software was used to match spots for intensity differences and predict BPH induced proteins.. The protein spots were categorized as BPH altered (increased or decreased in abundance) if protein abundance in a rice line increased or decreased with BPH infestation compared to mean control value. Abundance ratio (protein volume in infested plants/control plants) was compared with control at a time point to determine fold change in proteins. An arbitrary cutoff was used to express highly altered proteins [>1.5 (increased), <0.5 (decreased) or >0.5 and <1.5 (least altered)].
4.5. Protein Identification
The proteins spots from Coomassie Brilliant Blue (G-250) stained gels were manually excised using a sterilized scalpel and submitted to the Australian Proteome Analysis Facility (APAF) Macquarie University, Sydney, Australia [55
] for characterization. Protein samples were analyzed with matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry using a Micromass Tofspec time-of-flight mass spectrometer (Micromass, Manchester, UK) at APAF following standard procedures. If proteins could not be identified with MALDI-TOF, a further analysis was performed on Q-TOF LCMS. For protein identification, peak lists were used and peptide masses were searched against SWISS-PROT and NCBInr databases using the Mascot search engine [56
] supported by Matrix Science Ltd., London. In MS/MS Ion Search, following parameters were used for database queries on monoisotopic peptide masses using the Viridiplantae and Oryza sativa
as taxonomic categories; peptide mass tolerance of 150 ppm; fragment mass tolerance: ±0.6 Da; variable modifications: Oxidation (M), Propionamide (C); and the maximum number of missed tryptic cleavages, 1. Peptide masses that yielded a significant ion score (p
< 0.05) were considered positively identified.
4.6. Statistical Analysis
Data analysis was performed with Statistical Analysis Software (SAS) (Version 9.1) and JMP-IN (Version 5.1) (SAS Institute, Cary, NC, USA) using protein abundance values in control and BPH infested plants of three genotypes (wild type IR64 and two mutants) and compared at each respective time point. Protein abundance ratio in relation to each control group (IR64 or mutants) was calculated by dividing the spot abundance in the BPH infested plants by the mean spot abundance of the control plants and expressed as fold change with statistical significance at p-value lower than 0.05. A 2-way ANOVA was used to compare the protein abundance between IR64 and the mutants and the means were separated with the Tukey’s HSD multiple means comparison test (p
< 0.05). Ordination statistics were performed on protein abundance and genotypes to measure interactions between the BPH and rice proteins (Canoco V.4.5) [57
]. Initially, detrended correspondence analysis (DCA) was performed to measure eigenvector length of expressed proteins variables (control, infested) [26
]. Redundancy analyses (RDA) were performed and the significance of the first two axes, as well all four axes, were tested using a Monte Carlo test with 1000 permutations in reduced space. The reason RDA was chosen in this particular instance rather than another multivariate method, is that the variable data showed linear
responses as opposed to unimodal
responses. Multivariate biplots allow one to explore trends through numerical data analysis above and beyond simple hypothesis testing. Where relationships and covariation between variables is not evident with simple univariate statistics, multivariate methods clearly show the abundance of specific proteins as variables in relation to experimental factors. In this case it is clear that specific proteins covary with specific treatments, and the treatments themselves also show covariation.