Proteomic responses of host pigeonpea during interaction with Fusarium udum pathogen

doi:10.21203/rs.3.rs-4886996/v1

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Proteomic responses of host pigeonpea during interaction with Fusarium udum pathogen

https://doi.org/10.21203/rs.3.rs-4886996/v1

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In India, pigeonpea is a drought-tolerant crop that is susceptible to more than 100 diseases, including viruses, bacteria, nematodes, fungus, and phytoplasma. The restrictions of utilising chemical fungicides make managing soil-born diseases, like Fusarial wilts, difficult. While using resistant cultivars is preferable, the emergence of novel disease variations frequently poses a challenge. The objectives of this study are to investigate root proteome responses, discover host plant proteins for cultivars that are resistant, and comprehend the molecular mechanisms behind the interactions between F. udum and pigeonpea. Proteomics was used to examine genotypes of pigeonpea fusarium wilt-susceptible (ICP 2376) and resistant (ICP 9174). To make ingress easier, seedlings were removed, cleaned, and chopped off. Both genotypes underwent total protein extraction, and the pigeonpea root protein underwent two-dimensional gel electrophoresis. A combination search of PMF and MS/MS data against the taxonomy of Viridiplantae was used to identify proteins. The goal is to comprehend the molecular processes that control plant resistance and susceptibility. Twelve of the 141 differentially expressed protein locations in the interaction between F. udum and pigeonpea were satisfactorily characterised. Based on their biological roles, seven differentially expressed proteins were found in the resistant cultivar and categorised into seven functional groups. Proteome profiles provided insight into the defense mechanism of pigeonpea against Fusarium udum infection by identifying two proteins, R56 and S41, as components of the defense mechanism. Greater accumulation of R56 in resistant cultivars suggests F. udum-induced defense, whereas down-regulation of S41 in susceptible cultivars supports direct defense.

Resistant

Susceptible

Cultivars

Fungal infection

Differential expression

Pigeon peas [Cajanus cajan (L.) Millspaugh], is a perennial plant belonging into Fabaceae family that are always cultivated as an annual crop due to their short lifespan. It is one of the main legume crops farmed in tropical and subtropical regions of the world, are hardy, broadly adaptable, drought-tolerant, and frequently cross-pollinated (Hillocks et al. 2000). India is considered the native of pigeonpea because of the genetic diversity in the local germplasm and the presence of wild relatives in the country (Van der Maesen, 1980). Owing to its spectrum of maturity, it can adapt to a wide range of environments, which makes it an optimal crop for sustainable agriculture systems in rainfed areas. Pigeon peas are mostly produced and consumed in India, where dried seeds are used to make the popular dish "dhal." The crop is mostly planted in the Indian states of Maharashtra, Karnataka, Uttar Pradesh, Madhya Pradesh, and Gujarat, and the it is the second most crop grown and farmed crop after chickpea.

The primary constraints to increasing the crop's output are its vulnerability to pests, illnesses, and other physiological stresses. More than 100 diseases, including bacteria, viruses, nematodes, fungi, and phytoplasma, have been found to harm pigeonpea (Nene et al. 1989); however, only a small number of these infections are known to cause economic losses. Currently, Phytophthora blight, sterility mosaic, and Fusarium wilt are the diseases of significant economic relevance. Of them, F. udum caused wilt is thought to be a significant factor in the low productivity of pigeonpea production; in India alone, the annual output loss resulting from this disease was estimated to be around 97, 000 metric tonnes (Saxena et al. 2010).

The pathogen mainly inhabits the soil; wilting symptoms can occur at any point during a plant's growth, but sensitivity spikes around the time of flowers. Plants that are infected exhibit signs of slow chlorosis and eventual death. The condition is characterized by vascular discolouration and an upward-extending purple band on the stem. The sole methods for chemical control of soil-born diseases, including pigeonpea wilt, are biological control and the use of resistant varieties, which pose a significant barrier for management. Nevertheless, the process of creating resistant cultivars is laborious and time-consuming. For more than 20 years, "ICP 8863," one of the wilt-resistant cultivars created by ICRISAT, covered more than a million hectares of land in southern India where pulses were grown (Ravikumara et al. 2022). The resistant variety has recently demonstrated moderate to high levels of susceptibility in certain pockets of its cultivation. This can be the result of the emergence of novel F. udum variant or their predominance in different parts of India.

Nonetheless, the efficacy of resistant cultivars in disease control has been restricted due to the diversity of F. udum and the regular appearance of its infectious forms. Currently, nine variants are distributed in India; of these, variants 2 and 3 were found to be widespread and predominant in most pigeonpea producing regions, despite efforts to study the genetic diversity and aggressiveness of the pathogen populations (Kiprop et al. 2002; Reddy et al. 1998; Vishwa Dhar et al., 2011). According to Ravikimara et al. (2022) majority of the F. udum isolates were vulnerable to ICP 2376 and LRG 30, but host differentials ICP 9174, BDN-2, and Bahar (ICP 7197) were shown to be resistant.

A multitude of proteins are involved in the defensive response during plant-pathogen contact. It will be easier to comprehend host-pathogen interactions and host defense mechanisms if host plant proteins are characterised. A potent method for locating responsive proteins that might be implicated in mechanisms of stress tolerance or susceptibility is proteomic analysis of plants exposed to various stressful treatments. In addition, compared to bacterial and fungal foliar diseases, the very modest number of proteomic studies on plant-pathogen interactions has concentrated on root illnesses brought on by nematodes and soil-borne fungus (Houterman et al. 2007). Moreover, nothing is known about the proteome of pigeon pea roots or their response to fungal infections. Therefore, the goal of the current study was to identify host plant proteins that may be responsible for a resistant variety's ability to fend off the invading pathogen, investigate root proteome responses associated with the susceptible and resistance differentials of pigeonpea interaction with a virulent isolate of F. udum, and offer fresh perspectives on the molecular mechanism of plant-fungal interaction.

Plant growth and fungal treatment

Proteomics analysis was performed using genotypes of pigeonpea Fusarium wilt-susceptible (ICP 2376) and resistant (ICP 9174). Both genotypes of seeds were grown in a greenhouse maintained at 25 ± 2 ºC using polythene covers packed with sterilized river sand. Twenty to thirty seeds were put in each plastic bag and let to grow for eight days after the seeds were surface sterilised using two percent sodium hypochlorite for two minutes. The seeds were then rinsed in sterile water to remove the sodium hypochlorite. Using potato dextrose broth (PDB) and a shaker incubator set to 25 ± 1 ^oC for eight days with a 12-hour photoperiod, the pathogen (Fu-3) was mass multified. Using a hemocytometer, the conidial suspension of F. udum was diluted with distilled water to maintain the threshold level of inoculum (6 × 10⁶ conedia/ml). Thereafter, eight-day-old seedlings were gently removed from their polythene covers, any extra sand was cleaned off their roots by running tap water. Root tips approximately 0.5 cm was long was cutoff to allow the pathogen to enter the host, they were then dipped in the agitated inoculum suspension (6 × 10⁶ conedia/ml) for a duration of one to two minutes. After being inoculated, the seedlings were moved into 12-cm pre-irrigated pots, filled with sand and sterilised vertisol (3:1). Each pot had seven transplanted infected seedlings, with a minimum of three replications kept alive. As control samples, plants of both genotypes grown in soil devoid of inoculum were maintained. The infected and control plants were grown under identical conditions.

Total protein extraction

As treatment and control samples, both genotypes (ICP 9174 and ICP 2376) with and without F. udum inoculation were maintained. After 48- and 96-hours post-inoculation (hpi), root samples were collected, immediately frozen in liquid nitrogen, and kept at -80°C for additional examination. There were three biological replicates produced by repeating the full experiment, which involved fungal treatment and plant development. Proteins were extracted using sonication and the phenol-SDS buffer extraction technique from pooled tissue (Chatterjee et al. 2014). In summary, 1g of root tissue was homogenised in 3 ml of SDS buffer, which contained 30% sucrose, 2% SDS, 0.1 M Tris-Cl, 5% β-mercaptoethanol, and 1 mM PMSF (phenyl methyl sulfonyl fluoride), measured at pH 8.0. The extract was subjected to six rounds of sonication at 60 amps for 15 seconds each, followed by a treatment with tris-saturated phenol. At -20°C, four volumes of 0.1 M ammonium acetate in methanol were added to precipitate the phenolic phase overnight. The samples were then centrifuged for 20 minutes at 12,000 g for 4°C. The precipitate was then air-dried and rinsed three times in ice-cold acetone and ice-cold methanol. The final pellet was resuspended in 100 microlitres of sample buffer (pH range 4–7; GE Healthcare, Uppsala, Sweden) containing 7 M urea, 2 M thiourea, 2% CHAPS, 20 mM DTT, 0.5% IPG buffer, and 0.8% (v/v) immobilized pH gradient (IPG) buffer). and measured with the Bradford assay.

Two-dimensional gel electrophoresis (2D-GE)

An isoelectric focusing (IEF) strip with a pH range of 4–7 was loaded with pigeonpea root protein. IEF was programmed to run at 200 V for thirty minutes, 500 V for thirty minutes, 1000 V for thirty minutes, 1000–8000 V (gradient) for thirty minutes, and 8000 V for three and a half hours. Strips were equilibrated twice after IEF to reduce the protein, and then they were alkylated in the equilibration buffers with 2.5% (w/v) iodoacetamide and 2% (w/v) DTT for 15 minutes while gently rocking the strips at room temperature (25 ± 2°C). Second-dimension SDS-PAGE (12% vertical polyacrylamide slab gels) was used to separate the proteins, and modified Coomassie staining was applied to the gels. The 2-D gel images were scanned using GE Healthcare's image scanner, and they were examined using Image Master 2D Platinum v. 6.0 image analysis program (Amersham Biosciences). The average volume of every protein spot was determined by classifying the three images, which corresponded to three independent biological replicates for every treatment. Differentially accumulating proteins were defined as those with 1.5-fold variations (above or below) in protein abundance level with p < 0.05 (Rani and Podile 2014).

Protein identification using MALDI-TOF MS and MS/MS

Protein spots excised from 2D-PAGE gels were destained and gel digested according to the protocol (Shevchenko et al. 2006). Peptide sample (1 µl) was placed into an Anchor Chip MALDI Plate (Bruker Daltonics, Germany) along with the matrix (1 µl, α-cyano-4-hydroxy cinnamic acid, HCCA). Mass spectra were generated in an Autoflex II MALDI TOF/TOF (Bruker Daltonics, Germany) mass spectrometer equipped with a pulsed nitrogen laser (λ-337 nm, 50 Hz) in the m/z range from 500 to 3500 Da. To remove matrix peaks and tryptic autolysis peaks from the acquired spectra, Flex Analysis Software (version 2.4, Bruker Daltonics, Germany) was used. Peptide mass fingerprint (PMF) and MS/MS data were integrated and searched using the MS Biotools (version 3.2) software against the taxonomy of Viridiplantae (green plants) in the SwissProt and NCBInr databases using the MASCOT search engine (version 2.2). The resulting data allowed for the identification of proteins. Proteolytic enzyme (trypsin), global modification (caramidomethyl, Cys), variable modification (oxidation, Met), peptide charge state (1+), maximum missed cleavage of 1, and peptide mass tolerance (± 0.5 Da) were among the standard criteria utilized in the search. A maximum of 95% (p < = 0.05) was chosen as the significance threshold. The molecular weight search (MOWSE) score and the percentage of sequence coverage served as the acceptance criteria for protein identification. The strongest m/z readings from each sample were selected for further fragmentation (MS/MS). By selecting the decoy checkbox on the MASCOT search engine, an automatic decoy database search was carried out. By comparing peptides to a random sequence from a decoy database, a decoy search was carried out to prevent peptides from being mistakenly identified. Accepted results were limited to those with a false discovery rate of 0%. The final protein identification process involved using the MASCOT search engine to perform a combination search using PMF (peptide mass fingerprint) and MS/MS data.

The goal of studying the pigeonpea root proteome was to comprehend the molecular mechanism directing the pigeonpea plant's vulnerability and/or resistance to infection by F. udum fungal pathogen 48 and 96 hours after inoculation in the ICP 2376 and ICP 9174 cultivars. An average of 127 ± 20 distinct protein spots were resolved after the 2-DE gels were stained with Coomassie and the mascot program was used. Following protein spot image normalization and manual verification, differential spots with counts of 70 and 71 were found in resistant and susceptible cultivars, respectively. A spot was deemed variable if it met any of the following three requirements: (i) it had to be consistently present or absent in each of the three replicates; (ii) it had to show genotype- or treatment-ratio differences of at least 1.5; and (iii) statistically significant differences (P < 0.05) among genotypes or treatments.

Differential expression of the protein spots in resistant genotype (ICP 9174)

Following 48- and 96-hours following F. udum inoculation, a total of 70 differently expressed protein spots (R1-RR72) with a broad range of molecular weights (20.1 to 205.0 kDa) were seen in both inoculated and uninoculated plants (Fig. 1). All seventy differentially expressed protein spots were divided into six groups according to their molecular weights, which ranged from 14.3 to 205.0 kDa (Table 1). Group III has 33 distinct protein spots, while Group IV and Group V have 25 and 9 differential protein spots, respectively. In addition, all 70 protein spots were divided into three groups based on the pH range. Group I comprised fourteen proteins that were shown to be differently expressed and had a pH range of 4 to 5. In contrast, Group II had 35 differential proteins with a pH range of 5 to 6, while Group III included 21 differential spots with a pH range of 6 to 7 (Table 2). When compared to 48 and 96 hours after inoculation, Table 3 indicates that 44 differentially expressed proteins were down-regulated and 12 differentially expressed proteins were up-regulated. The first time point (48 hours after inoculation) showed an increase in two differentially expressed spots; however, 96 hours later, the same spots showed a decrease in expression. A distinct set of five differentially expressed protein spots (R21, R22, R50, R69, and R70) showed no change in volume inside the specific protein spot 48 hours after inoculation, however the identical set of proteins showed an increase in volume 96 hours later. In the resistant cultivar (IC9174) the unique protein spot R72 was expressed 96 hours after inoculation, but it was not present in the uninoculated condition (Fig. 3).

Table 1

Categorization of differentially expressed proteins in *Cajanus cajan* × *Fusarium udum* interaction based on molecular weight.
Group	Molecular weight (kDa)	ICP 9174		ICP 2376
Group	Molecular weight (kDa)	Total number of differentially expressed protein spots		Total number of differentially expressed protein spots
I	14.3 to 20.1	0	-	0	-
II	20.1 to 29.0	3	R59, R64, R65	3	S64, S65, S66.
III	29.0 to 43.0	32	R32, R33, R34, R35, R36, R37, R38, R39, R40, R41, R42, R44, R45, R46, R47, R48, R49, R50, R51, R52, R53, R54, R55, R56, R57, R58, R60, R61, R62, R63, R67, R71, R72	31	S37, S38, S39, S40, S41, S42, S43, S44, S45, S46, S47, S48, S49, S50, S51, S52, S53, S54, S55, S56, S57, S58, S59, S60, S61, S62, S63, S67, S69, S70, S71.
IV	43.0 to 66.0	25	R11, R12, R13, R14, R15, R16, R17, R18, R19, R20, R21, R22, R23, R24, R25, R26, R27, R28, R29, R30, R31, R43, R68, R69, R70	27	S10, S11, S12, S13, S15, S16, S17, S18, S19, S20, S21, S22, S23, S24, S25, S26, S27, S28, S29, S30, S31, S32, S33, S34, S35, S36, S68.
V	66.0 to 97.4	9	R1, R3, R4, R5, R6, R7, R8, R9, R10	9	S1, S2, S3, S4, S5, S6, S7, S8, S14
VI	97.4 to 205.0	0	-	0	-

Table 2

Categorization of differentially expressed proteins in *Cajanus cajan* × *Fusarium udum* interaction based on pH.
Group	pH range	ICP 9174		ICP 2376
Group	pH range	Total number of differentially expressed protein spots		Total number of differentially expressed protein spots
1	4–5	13	R9, R10, R18, R19, R20, R24, R26, R43, R44, R48, R49, R64, R71	7	S1, S17, S18, S36, S51, S55, S69
2	5–6	33	R1, R4, R5, R8, R12, R13, R14, R17, R21, R22, R28, R30, R32, R36, R37, R38, R39, R40, R41, R42, R47, R50, R58, R59, R60, R61, R62, R63, R65, R67, R69, R70, R72	31	S4, S5, S7, S10, S11, S12, S15, S16, S19, S20, S25, S26, S27, S37, S38, S39, S40, S41, S42, S43, S51, S54, S55, S56, S57, S58, S59, S63, S64, S67, S68
3	6–7	21	R3, R6, R7, R11, R15, R16, R29, R31, R33, R34, R35, R45, R46, R51, R52, R53, R54, R55, R56, R57 R68	35	S2, S3, S6, S8, S9, S13, S14, S21, S22, S23, S24, S28, S29, S30, S31, S32, S33, S34, S35, S44, S45, S46, S47, S48, S49, S50, S52, S53, S60, S61, S62, S65, S66, S70, S71

Table 3

Differentially expressed proteins spots in resistant ICP 9174 cultivar.
Sl. No.	Spot number	Differential Expression of Protein spots		Sl. No.	Spot number	Differential Expression of Protein spots
Sl. No.	Spot number	48 hpi	96 hpi	Sl. No.	Spot number	48 hpi	96 hpi
1	R1	↓	↓	36	R37	↓	↑
2	R3	↓	↓	37	R38	↑	↑
3	R4	↓	↓	38	R39	↓	↓
4	R5	↑	↑	39	R40	↑	↑
5	R6	↓	↓	40	R41	↑	↑
6	R7	↓	↓	41	R42	↓	↓
7	R8	↓	↓	42	R43	↓	↓
8	R9	↓	↓	43	R44	↓	↓
9	R10	↓	↓	44	R45	↓	↓
10	R11	↓	↑	45	R46	↑	↑
11	R12	↓	↑	46	R47	↑	↑
12	R13	↓	↓	47	R48	↓	↓
13	R14	↓	↓	48	R49	↓	↑
14	R15	↓	↓	49	R50	UC	↑
15	R16	↓	↓	50	R51	↓	↓
16	R17	↑	↓	51	R52	↓	↓
17	R18	↓	↑	52	R53	↓	↓
18	R19	↓	↓	53	R54	↓	↓
19	R20	↓	↓	54	R55	↑	↓
20	R21	UC	↑	55	R56	↑	↑
21	R22	UC	↑	56	R57	↓	↓
22	R23	↑	↑	57	R58	↑	↑
23	R24	↓	↓	58	R59	↓	↓
24	R25	↓	↓	59	R60	↑	↑
25	R26	↓	↓	60	R61	↓	↓
26	R27	↓	↓	61	R62	↑	↑
27	R28	↓	↓	62	R63	↓	↓
28	R29	↓	↓	63	R64	↓	↓
29	R30	↓	↓	64	R65	↓	↓
30	R31	↓	↓	65	R67	↑	↑
31	R32	↓	↓	66	R68	↓	↓
32	R33	↓	↓	67	R69	UC	↑
33	R34	↓	↓	68	R70	UC	↑
34	R35	↓	↓	69	R71	↑	↑
35	R36	↓	↓	70	R72	Absent	↑
Note: ↓ - Down regulated
↑ - Up regulated
UC- Un changed

Differential expression of the protein spots in susceptible cultivar (ICP 2376)

In both inoculated and uninoculated plants, a total of 71 differently expressed protein spots with a wide range of molecular weights (20.1 to 205.0 kDa) were seen 48 and 96 hours after F. udum was inoculation (Fig. 2). All the proteins that were differently expressed were divided into six groups according to their molecular weight, which ranged from 14.3 to 205.0 kDa (Table 1). Group II comprises three proteins, Group III consists thirty-one distinct protein spots, Group IV includes twenty-eight differential protein spots, and Group V had nine divergent protein spots. Three groups were formed from the 71 differently expressed protein locations based on their pH range. Group I consisted of seven differentially expressed proteins, with a pH range of 4 to 5. Group II included 31 differential proteins, with a pH range of 5 to 6, and Group III included the 35 differential spots, with a pH range of 6 to 7. (Table 2). At both time intervals (48 and 96 hours after inoculation), 34 differently expressed proteins were down-regulated and 25 differentially expressed proteins were up-regulated (Table 4). After 48 hours of inoculation, three distinct protein spots (S35, S43, and S57) showed decreased expression; nevertheless, during 96 hours, these same spots showed increased expression. Six differentially expressed locations were down-regulated after 96 hours of inoculation, despite being up-regulated at the first time point (48 hours). After 48 hours after inoculation, the overall volume of two differentially expressed protein spots (S10 and S61) remained unchanged, however after 96 hours, the same set of proteins showed an increase in volume (Fig. 3).

Table 4

Differentially expressed proteins spots in susceptible ICP 2376 cultivar.
Sl. No.	Spot number	Differential Expression of Protein spots		Sl. No.	Spot number	Differential Expression of Protein spots
Sl. No.	Spot number	48 hpi	96 hpi	Sl. No.	Spot number	48 hpi	96 hpi
1	S1	↓	↓	37	S37	↓	↓
2	S2	↑	↑	38	S38	↑	↑
3	S3	↑	↓	39	S39	↑	↑
4	S4	↑	↑	40	S40	↑	↓
5	S5	↓	↓	41	S41	↑	↓
6	S6	↑	↑	42	S42	↓	↓
7	S7	↑	↑	43	S43	↓	↑
8	S8	↑	↑	44	S44	↓	↓
9	S9	↓	↓	45	S45	↓	↓
10	S10	UC	↑	46	S46	↓	↓
11	S11	↑	↑	47	S47	↓	↓
12	S12	↑	↑	48	S48	↓	↓
13	S13	↑	↑	49	S49	↓	↓
14	S14	↓	↓	50	S50	↓	↓
15	S15	↓	↓	51	S51	↓	↓
16	S16	↑	↑	52	S52	↓	↓
17	S17	↑	↑	53	S53	↑	↓
18	S18	↓	↓	54	S54	↑	↓
19	S19	↑	↑	55	S55	↓	↓
20	S20	↑	↑	56	S56	↑	↑
21	S21	↓	↓	57	S57	↓	↑
22	S22	↓	↓	58	S58	↑	↑
23	S23	↓	↓	59	S59	↑	↑
24	S24	↑	↑	60	S60	↓	↓
25	S25	↑	↑	61	S61	UC	↑
26	S26	↑	↑	62	S62	↑	↑
27	S27	↑	↑	63	S63	↓	↓
28	S28	↓	↓	64	S64	↓	↓
29	S29	↑	↓	65	S65	↓	↓
30	S30	↑	↑	66	S66	↓	↓
31	S31	↓	↓	67	S67	↓	↓
32	S32	↓	↓	68	S68	↑	↑
33	S33	↓	↓	69	S69	↓	↓
34	S34	↓	↓	70	S70	UC	↓
35	S35	↓	↑	71	S71	↓	↓
36	S36
Note: ↓ - Down regulated
↑ - Up regulated
UC - Un changed

MALDI TOF MS/ MS for characterisation of the proteins involved in C. cajani × F. udum interaction

Within the interaction between F.udum and pigeonpea, 141 protein spots with variable expression were identified, originating from cultivars that were susceptible (71 spots) and resistant (70 spots). Twelve of the 141 protein spots with variable expression were effectively characterized utilizing the MALDI TOF MS/MS. Seven proteins were found to be differentially expressed in the resistant cultivar: ADP, ATP carrier protein (R16), phosphatidylinositol 4-Phosphate 5-Kinase (R53), glyceraldehyde-3-phosphate dehydrogenase (dependent on NADP) (R60), Camphene/Tricylene synthase, chloroplastic (R41), pathogenesis-related protein (R56), likely beta-1,3-galactosyl transferase 19 (R61), and one unidentified protein (I 40). However, five different proteins were found to be differentially expressed in the susceptible cultivar: Dirigent protein 2 (S51), Thaumatin-like protein (S41), Hypothetical protein (S4), ATP synthase D chain, mitochondrial (S67), and one protein associated with cilia and flagella (S50), which is thought to be related to the fungal (F. udum) cell wall. (Fig. 4a, b & c). Based on their presumed biological roles, the discovered proteins were divided into seven functional categories: proteins lacking a function were placed in the unclassified group (Table 5). Three proteins (S67, R16, and S4) were categorized under metabolism-related proteins. Proteins involved in biosynthetic processes (R41 and S51) and defense-related processes (R56 and S41) were assigned to two proteins each. In a similar vein, five individual proteins were assigned to five functional groups: development protein (R53), redox homeostasis protein (R60), protein modification (R61), metabolism-related protein (R16), and unclassified protein (R40). Nevertheless, S50 was also identified as a pathogen cell wall protein.

Table 5

Identified proteins and classification according to their functions.
Spot number	Protein name^a	Score	Species	Entry from NCBInr/ UniProt databases^b	Mr/pI (Theoretical)^c Experimental	PM^d	Coverage %	Functions
I. Metabolism related proteins
R16	ADP, ATP carrier protein, mitochondrial	43	Oryza sativa	ADT_ORYSJ	9.79	4	5	Catalyzes the exchange of ADP and ATP across the mitochondrial inner membrane
S67	ATP synthase D chain, mitochondrial	124	Arabidopsis thaliana	ATP5H_ARATH	5.09	3	16	Synthesis of ATP from ADP in the presence of a proton gradient across the membrane which is generated by electron transport complexes of the respiratory chain.
S4	Hypothetical protein	259	Phaseolus vulgaris	gi/593627323	5.77	6	15	ATP synthase beta subunit, nucleotide binding domain
II. Biosynthetic process protein
R41	Camphene/ Tricylene synthase, Chloroplastic	47	Solanum lycopercicum	TPS3_SOLLC	6.12	2	4	Monoterpene synthesis that catalyzes the formation of comphene and tricyclene from geranyl diphoshate
S51	Dirigent protein 2	69	Arabidopsis thaliana	DIR2_ARATH	8.94	1	6	Stereoselectivity on the phenoxy radical-coupling reaction and plays a central role in plant secondary metabolism
III. Defense related protein
R56	Pathogenesis- related protein	93	Phaseolus vulgaris	PR2_PHAVU	4.85	1	7	Defense response
S41	Protein P21 (Thaumatin like protein)	24	Glycine max	P21_SOYBN	4.84	1	4	Defense response
IV. Development protein
R53	Phosphatidylinositol 4- Phosphate 5- Kinase	35	Arabidopsis thaliana	P15K1_ARATH	9	1	2	Catalyzes the sysnthesis of phosphatidylinositol 4,5- bisphosphate and phosphatidy-inositol 3,4- bisphosphate
V. Redox Homeostasis
R60	NADP-dependent glyceraldehyde-3-phosphate dehydrogenase	34	Apium graveolens	GAPN_APIGR	7.49	1	4	Generating NADPH for biosynthetic reactions
VI. Signaling protein
R61	probable beta-1,3-galactosyltransferase 19	52	Solanum lycopercicum	gi\|460386112	6.7	1	2	Involved in the pathway protein glycosylation, which is part of protein modification
VII. Unclassified protein
R40	Unnamed protein product	52	Coffea canephora	gi/661898214	6.9	1	10	Unknown
S50	Cilia- and flagella-associated protein	61	Chlamydomonas reinhardtii	CFA54_CHLRE	7.82	3	1	Sub cellular movement
^Note:
^a Percentage of protein identity, species and UniProt accession number, where appropriate, from Blast comparison are displayed in brackets.
^b Experimental mass (Mr, kDa) and pI were calculated with PDQuest software (BioRad) and standard molecular mass markers. Theoretical values were retrieved from the protein database (NCBInr). The software assigns a standard spot number to each spot protein (SSP).
^c PM: number of peptides matched (from peptide mass fingerprinting) with the homologous protein from the database. Some of these peptides were automatically MSMS fragmented.
^d The significant (P < 0.05) changes (more/less abundant) are given as normalized volume (calculated with PDQuest software) ratios: SC (susceptible control, non-inoculated), RC (resistant control, non-inoculated), SI (susceptible inoculated) and RI (resistant inoculated). Single letters mean infected/control ratios. Superscript numbers (1, 2) represent hours after inoculation (24 and 72 hai, respectively). Gen

This is the first comparative proteomic study on pigeonpeas to identify the routes of resistance against F. udum infection. Understanding the interactions between F. udum and pigeonpea may also be greatly aided by the significance of these changes between the two genotypes varying in degrees of resistance. After 48- and 96-hours post-inoculation, 127 ± 20 protein spots were expressed in susceptible and resistant, inoculated cultivars. Due to genetic variations, differentiable patches were produced, which showed the inherent variance between susceptible and resistant genotypes (Chatterjee et al. 2014). Houterman et al. (2007) found 21 total proteins in the xylem sap of tomato plants infected with Fusarium oxysporum, while Chatterjee et al. (2014) identified 274 total proteins in samples of susceptible (FG 62) and resistant (WR 315) chickpea cultivars at 48, 72, and 96 hours after infection with F. oxysporum f. sp. ciceri, cause of wilt disease. Similar investigations into the interaction between chickpea and F. o. f. sp. ciceri were also carried out by Gupta et al. (2010) and Ashraf et al. (2009). Six groups of differentially expressed protein spots were identified based on molecular weight: 70 spots in resistant cultivars and 71 spots in susceptible cultivars. Three groups were created based on the pH range of the differentially expressed protein spots. Li et al. (2013) also noted that during the Fusarium-banana interaction, there were differential expressions of 27 protein spots in susceptible (cv Brazil), 16 from moderately resistant (cv Nongke No. 1), and 15 from resistant cultivar (Yueyoukang I). Previous studies on the relationship between Fusarium and wheat have been conducted by Wang et al. (2006) and Geddes et al. (2008). Furthermore, Rampitsch et al. (2006) revealed protein profiles in wheat rust, while Cao et al. (2008) observed changes in the root protein profiles of canola with club root disease. Differentially expressed proteins were found in the wheat spikelets of the resistant cultivar, "Ning7840" that were infected with F. graminearum (Zhou et al. 2005). In contrast, different expression patterns were observed in 150 protein spots within the mung bean yellow mosaic and Vigna mungo (Mung bean) interaction (Kundu et al. 2013).

Based on their presumed biological roles, the discovered proteins were divided into seven functional categories; proteins lacking a function were placed in the unclassified group. Three proteins (R16, S67, and S4) involved in catalysing the exchange of ADP and ATP across the mitochondrial inner membrane are related to energy and carbohydrate metabolism. When ATP synthase beta subunit, nucleotide binding domain, and respiratory chain electron transport complexes are present, a proton gradient across the membrane is created that facilitates the synthesis of ATP from ADP. In general, down-regulating the R16 and S67 protein sites resulted in a reduction in proteins and glucose metabolism. On the other hand, we noticed that the nucleotide binding domain (S4) protein of the ATP synthase beta subunit was upregulated. This outcome was comparable to the research on the interactions between F. oxysporum f.sp. pisi and Pisum sativum carried out by Castillejo et al. (2015). In a similar vein, Gupta et al. (2010) also noted that the proteins in charge of glucose metabolism expressed differently.

The pigeonpea infection caused by F. udum resulted in a down-regulation of R41 and S51 expression, which are proteins involved in the process of biosynthesis. These proteins are involved in the production of terpenoids, unsaturated fatty acids, lignan and lignin (Dirigent Protein 2), and camphene/tricyclene synthase and chloroplastic. These proteins stimulate the lignin-containing ferulic acid polymers that are attached to the cell wall in reaction, aiding in the plant's defense against diseases and damage. The non-oxydative phase of the pentose phosphate (PP) pathway includes enzymes called transaldolases, which are involved in lignification (Castillejo et al. 2015). It has been shown that in tomato plants infected with F. oxysporum, elongated papillae were generated as a result of continuous material deposition surrounding penetration hyphae. Both susceptible and resistant cultivars displayed the same frequency of hyphal formation after they were lignified, suggesting that they were successful in halting further hyphal growth (Bishop and Cooper, 1983a). In response to the pathogen attack, we observed that both susceptible and resistant cultivars in our system dramatically decreased their levels of proteins involved in the lignin-making process. It appears that the present findings contradict the findings of Castillejo et al. (2015). Reduced expression of the proteins involved in the biosynthetic processes has been demonstrated in a number of systems to be the cause of a decrease in the lignins apposition for cell wall reinforcement and papillae development within epidermal and cortical cells (Olivain and Alabouvette, 1999; Ouellette et al., 2002).

Furthermore, two proteins P21/Thaumatin-like protein (S41), and pathogenesis-related protein (R56) involved in the defense mechanism were found in both susceptible and resistant cultivars. These proteins were deployed in the defense against infections, pests, and herbivores. At both time intervals (48 and 96 hpi), there was a higher accumulation of R56 spot (pathogenesis-related protein) in the resistant cultivar (ICP 9174). But in the susceptible cultivar (ICP 2376), the accumulation of proteins P21/Thaumatin-like protein (S41) was shown to be increasing up until 48 hpi. However, after 96 hpi, the same protein abruptly dropped (down-regulated). Similar to this, PR1 (pathogenesis-related protein 1), BGL (glucan endo1-3 beta glucosidase), TLP (thaumatin-like protein), and TPI (trypsin protease inhibitor) were found to accumulate, according to Chatterjee et al. (2014). During defense, nonexpressor of PR genes (NPR1) is known to control PR1 expression (Aboul-Soud et al., 2009). Moreover, PR1 is positively regulated by ACD, which is known to hasten cell death in Arabidopsis (Lu et al. 2003). In addition to controlling PR1 expression, MAP kinase (mitogen activated protein kinase), EDS4 (increased disease susceptibility 4), and PAD2 (phytoalexin deficient 2) are associated with the fungal defense response (Quilis et al. 2008). The current investigation indicates that the upregulation of PR protein in resistant plants, plays a direct part in the defense generated by pigeonpea and F. udum and interaction.

Following pathogen inoculation at 48- and 96-hours post-inoculation, there was a reduced accumulation (down-regulation) of the sole development protein, R53 (Phosphatidylinositol 4-Phosphate 5-Kinase). Normal plant development and defense responses entail the production and catalysis of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 4,5-bisphosphate, which are catalysed by the same proteins. Similarly, extracellular manganese-SOD activity has been linked to development proteins such as germins, which are known to have roles in plant defense and development (Carter and Thornburg, 2000). Actin monomer-binding proteins called structural protein profiles (PRFs) control how uncapped actin monomers assemble and disassemble to create cytoskeleton filaments (Day et al. 2011). However, at 48 and 96 hpi, resistant cultivars showed an up-accumulation of the redox homeostasis protein (R60). The production of NADPH for biosynthetic reactions is a function of these glyceraldehyde-3-phosphate dehydrogenase proteins, which are dependent on NADP. Reactive oxygen species (ROS) are produced and accumulated in plants in response to pathogen infection; this defense mechanism is well-documented (Torres, 2010). According to Averyanov (2009), the oxidative burst is the first common event in plant-pathogen interactions. However, in F. oxysporum f. sp. pisi and Pisum sativum interactions, Castillejo et al. (2015) discovered that two redox homeostasis proteins (aldo/keto reductase and 12-oxophytodienoic acid 10, 11-reductase) increased in response to inoculation.

Once biotic and abiotic challenges are recognised at the cellular level, signal transduction pathways are triggered. This results in modifications to many metabolic pathways and cellular activities, including photosynthesis, cell rescue and defense mechanisms, and redox homeostasis. The F. udum infection causes a build-up of likely beta-1,3-galactosyltransferase 19 (R61), a component of the protein modification pathway that is involved in the glycosylation of proteins. In a similar vein, Gu et al. (1996) discovered that injury brought on by bacterial pathogen infection in tomatoes induces leucine aminopeptidase (LAP). While over expression of 14-3-3 promotes resistance and causes a hypersensitive plant response, lower expression of 14-3-3 in Arabidopsis plants impairs resistance to powdery mildew fungal infection (Yang et al. 2009). However, because of the up-accumulation of another unclassified protein (S50) and the down-regulation of one cilia- and flagella-associated protein (S50), which is thought to be a fungal cell wall-related protein involved in subcellular movement, the precise functions of the proteins are unknown. It is thought that these unclassified proteins will now have appropriate names and functional designations thanks to the recent release of the pigeonpea complete genome sequence and the update of functional annotations (Varshney et al. 2012).

In conclusion, this study enabled us to present evidence of proteomic changes caused by F. udum in susceptible and resistant pigeonpea varieties. This allowed us to identify important molecular elements of the particular host-pathogen interaction and may be used to create genetic markers that could be used to screen collections of pigeonpea germplasm for resistance.

DECLARATION OF COMPETING INTEREST

There is no conflict of interest, according to the authors.

Author Contribution

1) Bankapura Mariyappa Ravikumara (BMR): Visualization, Validation, Formal analysis, Investigation, Writing-Original Draft, 2) Manjunath Krishnappa Naik (MKN): Conceptualization, Writing-Review and Editing, Supervision, Mamta Sharma (MTS): 3) Conceptualization, Methodology, Editing 4) Swaroopa Rani T (SRT): Methodology, Investigation, 5) Pooja Bhatnagar Mathur (PBM): Methodology, Investigation, 6) Moka Rajasekhar (MRS): Review and Editing

Acknowledgement

Thanks are due to the University of Agricultural Sciences, Raichur for funds and facilities provided, and the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) for the fellowship as well as facilities. We also acknowledge Platform for Translational Research on Transgenic Crops (PTTC), ICRISAT, Hyderabad for enabling the proteomics. Finally highly thankful to MAHE, Manipal for providing facilities and conceptualization.

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No competing interests reported.

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Proteomic responses of host pigeonpea during interaction with Fusarium udum pathogen

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Abstract

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INTRODUCTION

MATERIALS AND METHODS

Plant growth and fungal treatment

Total protein extraction

Two-dimensional gel electrophoresis (2D-GE)

Protein identification using MALDI-TOF MS and MS/MS

RESULTS

Differential expression of the protein spots in resistant genotype (ICP 9174)

Differential expression of the protein spots in susceptible cultivar (ICP 2376)

DISCUSSION

Declarations

DECLARATION OF COMPETING INTEREST

Author Contribution

Acknowledgement

References

Additional Declarations

Status:

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