Expression profiling, biochemical and histochemical analysis of contrasting cultivars provide insight into resistance against white rust disease (Albugo candida) in Brassica juncea L

doi:10.21203/rs.3.rs-4260272/v1

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Expression profiling, biochemical and histochemical analysis of contrasting cultivars provide insight into resistance against white rust disease (Albugo candida) in Brassica juncea L

https://doi.org/10.21203/rs.3.rs-4260272/v1

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Background

White rust disease caused by the biotrophic oomycete Albugo candida is one of the most serious impediments in realizing the production potential of Brassica juncea. Moreover, due to the obligate nature of the pathogen, R-gene-based resistance is unstable as the newer virulent races emerge quickly. Therefore, a deeper understanding of the molecular basis of resistance is essential for developing durable resistant varieties. In this study, we selected susceptible cultivar, ‘Pusa Jaikisan’ and its single R-gene-based resistant NIL, ‘Pusa Jaikisan WRR’ for elucidating the defense mechanism in B. juncea against A. candida.

Results

Comparative histochemical analysis at 12 dpi showed higher callose deposition in the resistant cultivar than in the susceptible cultivar which hints towards its possible role in defense mechanism. Based on the biochemical markers observation, total protein was found to have a negative correlation with the resistance. The antioxidant enzymes (POX, CAT, and SOD) and non-enzymatic ROS scavenging compounds such as polyphenols and proline showed a positive correlation with the white rust resistance. The PPO, total chlorophyll and total carotenoids were also found to show higher activity in the ‘Pusa Jaikisan WRR’. According to the heat map analysis, PAL was identified to be the most induced enzyme involved in the defense mechanism. Furthermore, the expression analyses of defense related markers such as salicylic acid (SA) associated PR protein genes (PR1 and PR2) and jasmonic acid (JA) associated PR protein genes (PR3 and PR12) were done by qRT-PCR. Based on the results, PR2 emerged as the best possible gene for defense against A. candida followed by PR1. PR3 and PR12 also showed positive correlation with the disease resistance which may be due to the JA pathway acting complementary to the SA pathway, thus indicating a synergistic JA-SA hormonal crosstalk in case of B. juncea-A. candida interaction.

Conclusion

The present study establishes a major role of simulated response of the defense molecules which can stop the disease progression thus incurring resistance. This may be used in the future for developing resistance against the biotrophic pathogen especially A. candida in B. juncea.

Albugo candida

biotrophic

Brassica juncea

PR protein genes

biochemical

ROS

callose

Oilseeds are one of the economically significant crops next to cereals. The global trends of area, production, and yield for oilseed production in the year 2021-22 were 41.95 mha, 88.35 mt and 2110 kg/ha, respectively. In India, rapeseed mustard has a share of one-third of total edible oil production and one-fourth of the total cultivated area. Out of this, Indian mustard [Brassica juncea (L.) Czern. And Coss.] has the largest share (about 36%) in the India’s edible oil reservoir (ICAR-DRMR, 2023). Despite this, India imported 13.11 lakh tonnes of edible oil in June 2023, which is alarming (The Solvent Extractors’ Association of India, 2023). Many biotic and abiotic stresses are serious impediments in realizing the potential production of B. juncea. Among all these, white rust disease caused by Albugo candida is globally a serious problem. It is known to cause losses ranging from 1–90% which depends on a multitude of factors such as plant population, host genotype, nutrition, and congenial environment for disease progression (1). Chemicals such as Mancozeb and a combination of metalaxyl and mancozeb have proven to be a quick and effective method of disease management but they come with their own set of economic and environmental antagonistic spillovers (2). Therefore, exploring inherent resistance and breeding for it, is the best strategy for managing this disease (3).

The plant defense system works along multiple lines of action. Presence of pathogen or pathogen sensing on plants is aided by pathogen-associated molecular pattern (PAMP) which are recognized by host cell surface receptors called pathogen recognition receptors or PRRs, resulting in PAMP triggered immunity (PTI) (4). This trigger signal transduction cascades resulting in an immediate and broad-spectrum defense response achieved through increased expression and activation of antioxidative enzymes and defense enzymes, such as catalase, peroxidase, polyphenol oxidase, phenylalanine ammonia lyase; pathogenesis-related proteins such as β,1–3, glucanase, and chitinase; and phenolics and other antimicrobial compounds such as defensin and phytoalexin (5, 6). PTI is followed by effecter triggered immunity (ETI) that occurs in the cytoplasm and is associated with the R-gene based immunity (7). However, A. candida being an obligate biotroph shares a strong co-evolutionary relationship with the host which enables quick racial evolution thus breaking down the already existing R-gene-based resistance, posing an additional threat to the brassica cultivation. Given these factors, genetic engineering for the broad-spectrum non-host resistance (NHR) genes is the most apt method for managing the dreaded white rust (8, 9).

Each successful disease establishment involves a plethora of molecular and biochemical events between the host plant and its pathogen such as production of PR proteins, ROS, cell wall reinforcement and antimicrobial compounds produced through the mitogen-activated protein kinases (MAPKs) mediated signaling pathways (10). Cell death caused by ROS is usually favoured by the pathogen in plant-necrotroph interaction but is an important component of disease resistance in the case of plant-biotroph interaction (11, 12). At the same time, it needs to come down as excessive cell death can be detrimental for the plant itself. Hydrogen peroxide (H₂O₂) is one of the most important components of ROS and accumulates after the plant recognizes the pathogen. Hydrogen peroxide is converted into water and oxygen in peroxisome by catalase (CAT) (EC 1.11.1.6), whereas peroxidases (POX) (EC 1.11.1.7) reduce H₂O₂ along with many phenolics and non-phenolics substrates. Superoxide dismutase (SOD) (EC 1.15.1.1) negates the harmful effect of superoxide radicals by dismutation into oxygen and water (13, 14). The polyphenols play an important role by neutralizing the ROS, while polyphenol oxidase (PPO) (EC 1.10.3.2 or EC 1.14.18.1) catalyses the oxygen-dependent oxidation of polyphenols to quinones. Together these two maintain ROS-homeostasis, which helps in plant defense (15, 16). Proline is an important non-enzymatic antioxidant that can also neutralize ROS and act as a balancing wheel (17). Phenylalanine ammonia-lyase (PAL) (EC 4.3.1.24) is one of the most crucial enzymes in the defense pathway. It catalyses the deamination process of phenylalanine into secondary phenylpropanoid metabolism (18). For producing these enzymes, the protein is broken down hence decreasing its concentration upon resistance activation (19). The relationship between chlorophyll and carotenoid with resistance is not strongly ascertained and needs future studies.

PR proteins along with hormonal responses induced by complex signalling pathways are the major determinants of plant resistance and are strongly induced upon pathogen inoculation (8, 20). These are small proteins which are accumulated and induced in response to an array of biotic and abiotic stresses (21). At present, PR proteins are classified into 17 families and among them, the major ones are PR1 (antifungal), PR2 (β-1,3-glucanase), PR3 (chitinase), PR5 (thaumatin-like protein), PR9 (peroxidases), PR12 (plant defensin), PR13 (thionins) (22). Regarding hormonal signalling-mediated resistance, salicylic acid (SA) and jasmonic acid/ethylene (JA/ET) are important components. They are thought to be mutually antagonistic. The JA/ET pathway is initiated upon attack by a necrotroph while in the case of a biotroph, the SA pathway is predominant. Multiple reports hint at a positive role of JA/ET in the case of biotroph attacks where they complement the SA pathway (23). PR1, PR2 and PR5 are the SA-marker genes, while PR3, PR12 and PR13 are the JA/ET-marker genes (24). As, B. juncea suffers from a heterogeneous group of pathogens which includes both biotrophs and necrotrophs, the signalling pathway genes may help in developing broad-spectrum durable resistance. Several transgenic plants over-expressing PR genes have been developed against B. juncea pathogens imparting disease resistance (25, 26).

In present study a comparative analysis was done between the two B. juncea genotypes, R-gene-based NIL- ‘Pusa Jaikisan WRR' and the susceptible ‘Pusa Jaikisan’ to better understand the resistance mechanism against A. candida and studied the disease progression, callose deposition after infection, defense-related enzymes and non-enzymatic compounds, and the role of PR genes as markers of the hormonal signalling pathway in resistance (27). Based on the morphological differences of symptom development on leaves of these two varieties, specific time frame was chosen, 0, 12, 24, 48, 72 and 96 hpi, to assess the differences in host response. Overall, this study is focused on morphological symptom development after pathogen inoculation, followed by assessment of the defense enzymes and PR proteins biochemically, and through gene expression levels in the two genotypes.

Plant materials and fungal materials

Two cultivars of Indian mustard (B. juncea), ‘Pusa Jaikisan’ and ‘Pusa Jaikisan WRR’ were used as plant materials and the seeds were obtained from the ICAR-Directorate of Rapeseed Mustard Research. These experiments were carried out from November 2022 to March 2023 at the Division of Plant Pathology, ICAR-Indian Agricultural Research Institute. The research complied with the institutional, national, and international guidelines and legislation. Albugo candida infecting ‘Pusa Jaikisan’ (B. juncea) was collected from the field of Division of Genetics, ICAR-IARI, New Delhi (isolate Ac-Dli). Furthermore, the isolate was purified and maintained on seedlings of ‘Pusa Jaikisan’.

Inoculation test

Seeds of ‘Pusa Jaikisan’ and ‘Pusa Jaikisan WRR’ were sown in trays in three replications and were kept under 16 h light alternated with 8 h dark at 21⁰C. Seven-day-old plants were spray inoculated with the Ac-Dli inoculum (concentration ~ 1x 10⁵ spores/ml). The plants were then maintained in a dark growth chamber for 24 h at 22 ⁰C with 100% humidity. The plants were then moved into a growth chamber and provided controlled conditions for 16 h light and 8 h dark at 21 ⁰C, with regular irrigation. After 12 days, disease observations were made. The infected leaf samples were harvested from ‘Pusa Jaikisan’ and ‘Pusa Jaikisan WRR’ at 12, 24, 48, 72 and 96 hpi, snap freeze in liquid nitrogen and stored at -80 ⁰C for further analyses.

Disease parameters

Disease resistance or susceptibility were assayed on the leaves of the three plants at 12 dpi. White rust disease scoring scale of 0 to 9 was used to characterize the plants in which 0, 1, 3, 5, 7 and 9 correspond to 0, < 5, 5–10, 11–25, 26–50 and > 50% diseased area respectively (28). The diseased area comprised white-coloured zoosporangial pustules. The experiment was carried out in three replications and for a single replication, five leaves were taken from each plant.

Confocal microscopy

Callose deposition near plasmodesmata was observed following the (29). For sample preparation and tissue staining, the leaf tissues were collected at 12 dpi from both ‘Pusa Jaikisan’ and ‘Pusa Jaikisan WRR’. The entire leaf was cut along the petiole and submerged in 96% ethanol solution overnight for destaining. The destained leaves were rinsed with distilled water to remove excess ethanol from the surface. Using a sharp razor blades 5 mm strips of leaves were cut ensuring that sample were taken from the same region in both susceptible and resistant leaves as there might be differences in callose deposition depending on the area. The sample was then rehydrated by sinking the strips in double distilled water (DDW) followed by incubation at room temperature (RT) for 1h. The strips were removed from DDW and placed in a 25 ml glass tube filled up to 1/3rd with aniline blue solution. The vacuum desiccator was used for 10 min to help dye penetrate the leaf tissues. Tubes were now wrapped with aluminum foil and incubated at RT for 2h. Specimens were then taken for microscopy and were mounted on microscopic slides and observed under a confocal microscope at ICAR-IARI, New Delhi (Model: Leica TCS SP52). Configuration of confocal microscopy was built in a single-track mode. The 405 nm diode laser was used for excitation, while the 475–525 nm band passed emission filter for the aniline blue fluorescence. A white pseudo colour was used for aniline blue emission. The important area under the microscope showing disease progression was captured by a camera mounted on the system and photomicrographs were processed in Image J software.

Biochemical assays

The biochemical assay was carried out from the infected leaf samples at 0, 12, 24, 48, 72 and 96 hpi. For the enzyme extract, 200 mg of leaf tissue was crushed into a fine powder using liquid nitrogen followed by homogenizing in 1.5 ml ice cold 50mM Sodium phosphate buffer (pH 7.0), 2mM EDTA, 5mM β-mercaptoethanol and 4% Polyvinylpyrrolidone (PVP). The homogenate was centrifuged at 3000 rpm for 30 min at 4 ⁰C. The supernatant was stored in aliquots at -40 ⁰C for the estimation of total protein, polyphenols, PPO, PAL, CAT, SOD and POX.

Total Protein estimation

The total protein content was determined using Bradford’s reagent (1976), keeping the bovine serum albumin as the standard. For the assay, 2 ml Bradford reagent was added in a test tube along with 990 µl water and 10 µl enzyme extract. The absorbance was recorded at 595 nm by using a UV-visible spectrophotometer. Total protein content was expressed as mg/g dry weight.

Peroxidase (POX) assay

For determining POX activity (µmol tetra guaiacol min^-1 mg^-1 protein), (30) method was followed. Three solutions, solution A containing Sodium phosphate buffer (100mM, pH 7.0), solution B containing 10 mM H₂O_2, and solution C made of 20mM guaiacols were freshly prepared. In a cuvette, 1.5 ml Solution A and 0.12 ml Solution B were mixed. Absorbance increment was recorded at 470 nm wavelength for 3 min at intervals of 1 min each. The reaction mixture without enzyme served as blank. Tetra guaiacol (26.6 mM^-1cm^-1) was used as the extinction coefficient of the oxidation product.

Catalase (CAT) assay

Methodology given by (31) was used for the estimation of CAT activity (H₂O₂ reduced min^-1 mg^-1 protein), where solution A: Sodium phosphate buffer (100mM, pH 7.0) and solution B: 30 mM H₂O₂ were freshly prepared. 0.1 ml enzyme extract was mixed with 1.5 ml solution A and 0.4 ml distilled water. The decrease in absorbance was recorded for 3 min at 30 s intervals at 240 nm just after the addition of 1ml solution B. The extinction coefficient of H₂O₂ (39.4 mM^-1 cm^-1) was used for quantification.

Superoxide dismutase (SOD) assay

SOD activity (µmol min^-1 mg^-1 protein) was estimated by (32) methodology. The assay solution comprised 27 ml-50mM Sodium phosphate buffer (pH 7.8), 1 ml-1.72 mM Nitroblue tetrazolium (NBT), 1.5 ml-201 mM solution and 0.75 ml-1% Triton X-100. To determine the enzymatic activity, 1 ml solution mix, 0.1 ml enzyme extract and 0.03 ml riboflavin were mixed in a test tube. They were kept in a foil-lined box with an internally mounted source of illumination viz., a 40W fluorescent bulb for 10 min for incubation. The reaction was terminated by switching off the bulb and observing the absorbance at 560 nm. A reaction without enzyme extract was taken as 100% NBT reduction or no inhibition and it served as the blank.

Total Polyphenols

Total polyphenol content was determined by (33). A diluted test sample (0.5 ml) was reacted with 0.2 ml Folin-Ciocalteau reagent, and the reaction was neutralised with sodium carbonate (0.5 ml). 10 ml distilled water was used to make up the volume. The absorbance of the blue colour was measured at 760 nm. The gallic acid standard curve was used for quantification.

Polyphenol oxidase (PPO) activity

PPO activity was measured following (34) methodology. Fresh leaves (0.2 g) were homogenized in 2 ml of pH 6.5 0.1 M sodium phosphate buffer and centrifuged at 16,000 rpm for 15 min at 4⁰C. The enzyme was obtained from the supernatant. 200 µl of enzyme extract and 1.5 ml of 0.1 phosphate buffer (pH 6.5) were used for the process. The reaction was started by using 200 µl of 0.01 M catechol, and the enzyme activity was measured at 495 nm/min/mg protein.

Phenylalanine ammonia lyase (PAL) assay

PAL activity (µmol trans-cinnamic acid min^-1 mg-¹ protein) was measured by (35) methodology. 25 mM tris buffer (pH 8.8) was used as the homogenisation buffer. After adding 0.1 ml of enzyme extract and 0.4 ml of 0.05 M tris HCL buffer (PH 8.8) having 0.2 mM phenylalanine, the reaction mixture was incubated in a water bath at 37⁰C for 60 min. To stop the reaction, 0.1 ml of 0.5 N HCL was added. 2 ml of toluene was used to extract the trans-cinnamic acid. The absorbance was measured at 412 nm followed by calculating enzymatic activity.

Total proline test

The free proline content was calculated by (36) methodology. A 0.2 g of fresh leaf sample was homogenised in 2 ml of 3% w/v distilled water sulfosalicylic acid and centrifuged at 10,000 rpm for 30 min at 4 ⁰C. 1 ml of supernatant was combined with 1 ml of glacial acetic acid and 1 ml of acid ninhydrin reagent and incubated for 1 hour at 100 ⁰C in a water bath before being immediately plunged into an ice bath to stop the reaction. 2 ml of toluene was after that transferred to each reaction mixture tube. The chromophore was finally extracted by vigorously swirling on a vortex mixer. A spectrophotometer was used to measure the absorbance of the chromophore-comprising toluene layer at 520 nm.

Chlorophyll and carotenoid content

Chlorophyll content (mg/g fresh weight) was calculated by placing the 10 mg of fresh tissue in a 2 ml microcentrifuge tube and 700 µl of pre-heated DMSO (dimethyl sulphoxide) was added to the tube containing the leaf tissue. For 30 min, the tube was placed in a 65 ⁰C water bath. Following incubation, 300 µl of DMSO was added to achieve a volume of 1 ml. The absorbance was measured at three different wavelengths: 470 nm, 645 nm, and 663 nm. Arnon’s equations were used to calculate the chlorophyll content (37). (38) methodology was used to determine the total carotenoid content (mg/g fresh weight) by using the same enzyme extract.

Gene expression profiling

For the gene expression profiling, RNA was isolated from the infected leaf samples at 0, 12, 24, 48, 72 and 96 hpi using RNeasy® Plant Mini Kit QIAGEN (Germany) as described by the manufacturer’s protocol. The purity and concentration of RNA was determined using Nanodrop (Thermo Scientific, Wilmington, DE). The quantified RNA was then used to synthesize first-strand cDNA through Promega ImProm-IITM Reverse Transcription system (USA) following the manufacturer’s protocol. For designing the primers from conserved regions of PR1, PR2, PR3 and PR12 genes, oligoanalyzer software was used (Table 1). For qRT-PCR reaction, 2 µl of cDNA, 5 µl of SYBR green qRT-PCR master mix (Takara kit, Japan) and 0.5 µl of each primer was mixed. The reactions were performed in triplicates and repeated using three biological replicates, keeping Alpha-tubulin (housekeeping gene) as an internal control. The thermal conditions were kept at 94 ⁰C for 3 min, followed by 40 cycles of 94 ⁰C for 30 s, 60 ⁰C for 30 s, and 72 ⁰C for 30 s. Relative expression levels of each gene were calculated using C_T method (39). Fold changes with p ≤ 0.05 were taken as the significant level of expression.

Table 1

List of Primers used for the qRT-PCR analysis of PR1, PR2, PR3 and PR12 genes at 0, 12, 24, 48, 72 and 96 hpi. These primers are derived from the conserved region of *B. juncea.*
S. No.	Primer name	Primer sequence
1.	PR1-For	5’-GAACACGTGCAATGGAGAATG-3’
2.	PR1-Rev	5’-CCATTGTTACACCTCGCTTTG-3’
3	PR2-For	5’-CGTCTCTCTACAATTCGCTCTG-3’
4.	PR2-Rev	5’-CGATATTGGCGTCGAATAGGT-3’
5.	PR3-For	5’-AAGACCAGGTTCTTGCCTTC-3’
6.	PR3-Rev	5’-TCCGGTACACTCCCTACTATTC-3’
7.	PR12-For	5’-CAATGGTGAAAGCGCAGAAG-3’
8.	PR12-Rev	5’-AGGTGATGCACTGGTTCTT-3’
9.	Alpha-tubulin-For	5’-GCCTCGTCTCTCAGGTTATTTC-3’
10.	Alpha-tubulin-Rev	5’-TGAAGTGGATTCTTGGGTATGG-3’

Statistical analyses

A complete randomized design was set up for the experiment and the disease was scored by taking three replicates from each infected plant. The results were analysed by using the one-way analysis of variance (ANOVA) p ≤ 0.05 level of significance through Microsoft Excel.

Phenotypic disease assessment

The disease response of ‘Pusa Jaikisan’ and 'Pusa Jaikisan WRR’ was recorded on the basis of leaf area covered by pustules on the abaxial surface of the cotyledonary leaves at 12 dpi. The control genotype ‘Pusa Jaikisan’ was found to be highly susceptible with more than 50% area covered by the creamy white sporangial mass (pustules) and initiation of chlorosis, while ‘Pusa Jaikisan WRR’ was found to be immune with no pustules and 0% leaf area covered. Therefore, based on the 0–9 disease rating scale, the genotypes ‘Pusa Jaikisan WRR’ and ‘Pusa Jaikisan’ were rated 0 and 9, respectively (Fig. 1) (Khajan et al., 2018).

Comparative callose deposition

Callose deposition is an important determinant of resistance and was histochemically analysed through aniline blue staining. Following inoculation, leaf sample from both the specimens i.e., infected ‘Pusa Jaikisan’ and ‘Pusa Jaikisan WRR’ was withdrawn at 12 dpi and observed for callose deposition under confocal microscope. The callose was mostly deposited at plasmodesmata, between the plant cell wall and plasma membrane, and on other plant tissues. The callose deposition was observed at different scales i.e., 250 µm and 100 µm for ‘Pusa Jaikisan WRR’ and 250 µm and 75 µm in the case of ‘Pusa Jaikisan’. The quantum of callose deposition at the site of pathogen intrusion was found to be way higher in the case of ‘Pusa Jaikisan WRR’ as compared to ‘Pusa Jaikisan’ (Fig. 2). This hinted towards its possible role in the defense against white rust infection.

Comparative estimation of defense-related biochemical parameters

The comparative biochemical characterization between ‘Pusa Jaikisan WRR’ and 'Pusa Jaikisan’ at 0, 12, 24, 48, 72 and 96 hpi showed higher accumulation of different defense related biochemical compounds (POX, CAT, SOD, PAL, PPO, Polyphenols, Proline) in ‘Pusa Jaikisan WRR’ except proteins.

The total protein content was found comparatively higher in ‘Pusa Jaikisan’ at all the time points post inoculation (Fig. 3a). However, its concentration significantly dropped in both the genotypes as disease progressed. While it decreased till 48 hpi in ‘Pusa Jaikisan WRR’, in case of ‘Pusa Jaikisan’ the drop was observed up to 72 hpi, followed by a slight increase at 96 hpi.

In case of peroxidase test, a gradual increase in its activity was observed in ‘Pusa Jaikisan’, however, for ‘Pusa Jaikisan WRR’, its peak activity was found at 48 hpi followed by decrease thereafter (Fig. 3b). Both the CAT and SOD activity was found to be significantly higher in the ‘Pusa Jaikisan WRR’ as compared to ‘Pusa Jaikisan’ at all time intervals. While, in the case of ‘Pusa Jaikisan WRR’, CAT activity rapidly rose to the highest at 48 hpi and gradually decreased thereafter, a steady rise was observed in its activity for ‘Pusa Jaikisan’ (Fig. 3c, Fig. 3d). PAL activity was recorded to be higher in the ‘Pusa Jaikisan WRR’ as compared to ‘Pusa Jaikisan” at all the time intervals. The enzyme activity was found to be maximum at 72 hpi in the case of ‘Pusa Jaikisan WRR’ and decreased at 96 hpi. However, a steady rise in the activity of PAL was seen in ‘Pusa Jaikisan’ (Fig. 3e). The PPO was recorded to be highest in ‘Pusa Jaikisan WRR’ at 12, 24 and 48 hpi, whereas it was slightly higher in ‘Pusa Jaikisan’ at 72 hpi and 96 hpi than the former. The maximum PPO activity in ‘Pusa Jaikisan WRR’ was shown at 48 hpi, and a steady decline was seen thereafter. It kept on increasing across all the time points in ‘Pusa Jaikisan’ (Fig. 3f). In the case of total polyphenol, the activity was found higher in the ‘Pusa Jaikisan WRR’ as compared to the ‘Pusa Jaikisan’ at all the time intervals. The activity was found to be maximum at 72 hpi in the case of ‘Pusa Jaikisan WRR’ and it decreased at 96 hpi. There was a steady rise in ‘Pusa Jaikisan across all the time points (Fig. 3g).

The proline content was found to be higher in the ‘Pusa Jaikisan WRR’ as compared to the ‘Pusa Jaikisan’. There was an established difference in the enzyme activity of both genotypes at 0hpi. In the case of ‘Pusa Jaikisan WRR’, the maximum value was attained at 48 hpi followed by a steady decline. It kept on increasing till 72 hpi in ‘Pusa Jaikisan’ and there was a slight decline at 96 hpi (Fig. 3h). Total chlorophyll content was found to be higher in ‘Pusa Jaikisan WRR’ than in ‘Pusa Jaikisan’ at all the time intervals. In the case of Pusa Jaikisan’, the activity increased up to 48 hpi and then decreased from 72 hpi, whereas there was an increment up to 48 hpi followed by a decline at 72 hpi and then again, an increase at 96 hpi in the case of ‘Pusa Jaikisan WRR’ (Fig. 3i). The total carotenoid content was recorded to be higher in ‘Pusa Jaikisan WRR’ as compared to the ‘Pusa Jaikisan’ at all the time intervals. In the case of ‘Pusa Jaikisan’, it kept on increasing across the time intervals, whereas in ‘Pusa Jaikisan WRR’ it attained the peak value at 72 hpi and decreased thereafter (Fig. 3j).

Heat map analysis was done for different time intervals against 0 hpi to determine the enzyme with the highest response and best time interval for resistance induction (Fig. 4). Among all the biochemical markers, SOD, PAL, total polyphenols, and total carotenoids showed a higher quantum of jump as compared to the others. Most of the markers had their highest activity at 48 hpi or 72 hpi suggesting a strong defense response at these time intervals.

Comparative PR gene expression

PR proteins are an important part of the plant’s stress response machinery. PR1 and PR2 are the SA pathway genes pivotal in defense against the Biotrophs andPR3 and PR12 are JA/ET pathway genes, involved in defense against Necrotrophs. A higher expression of the PR1 gene was found in the ‘Pusa Jaikisan WRR’ as compared to the ‘Pusa Jaikisan’ at all time intervals. The maximum expression of PR1 (4.24-fold) was observed at 48 hpi, in ‘Pusa Jaikisan WRR’ thereafter a gradual decrease was observed (2.97-fold at 72 hpi and 2.84-fold at 96 hpi). Nearly similar expression was observed in ‘Pusa Jaikisan’ ranging from 1.41-fold to 1.63-fold (Figure 5a). In the case of PR2 gène expression, ‘Pusa Jaikisan WRR’ showed a higher expression at all time intervals except at 12 hpi as compared to the ‘Pusa Jaikisan’. The highest expression in ‘Pusa Jaikisan WRR was observed at 72 hpi (7.82-fold) which decreased to 5.66-fold at 96 hpi. In Pusa Jaikisan, no significant change in expression of PR2 and the maximum expression occurred at 24 hpi (Figure 5b). An interesting trend in the JA/ET-marker PR3 gene expression was observed, where the expression was higher in ‘Pusa Jaikisan WRR’ at 12, 24 and 72 hpi, whereas it was reversed at 48 and 96 hpi. In the case of ‘Pusa Jaikisan WRR’, maximum expression of PR3 was seen at 12 hpi (3.49-fold) and 24 hpi (2.84-fold), while for ‘Pusa Jaikisan maximum expression occurred at 48 hpi (2.84-fold). At 96 hpi, the expression was similar in both cultivars (Figure 5c). The expression pattern of PR12 did not reveal much fluctuation in ‘Pusa Jaikisan ’and ranged around 1.32-fold to 1.62-fold. In ‘Pusa Jaikisan WRR’, the highest expression (2.53-fold) occurred at 48 hpi. The resistant cultivar had higher expression at all time intervals except at 12 hpi. Both the cultivars had at par expression at 96 hpi (Figure 5d). The findings of this study hint towards a positive role of PR1 and PR2 against A. candida. Also, the higher expression of PR3 in the initial stages and higher expression of PR12 in ‘Pusa Jaikisan WRR may be due to their complementary role in resistance to the SA-marker genes. This suggests a possible synergistic role of both SA and JA/ET signalling pathways in white rust disease resistance. Further, Heat map analysis was done for different time intervals against 0 hpi to determine the PR gene with the highest response and best time interval for resistance induction (Figure 6). Among all the four genes, PR2 showed the highest relative expression. As with the biochemical markers, gene expression analysis also showed that 48 hpi and 72 hpi are the most active time intervals for defense-response.

The results from present study based on the histological, biochemical, and molecular alterations clearly showed that, among the two genotypes of Brassica juncea, ‘Pusa Jaikisan’ and ‘Pusa Jaikisan WRR’, the overall defense response was more pronounced and consistent in the R-gene based NIL ‘Pusa Jaikisan WRR’. The phenotypic disease expression under artificial conditions against A. candida showed vigorous development of infection in ‘Pusa Jaikisan’ with more than 50% of leaf area covered with creamy white pustules thus showing a highly susceptible reaction, however, no infection and pustule formation was observed in case of ‘Pusa Jaikisan WRR’ thus showing an immune response against this disease.

For any plant one of the earliest defense responses against haustorium-forming pathogens is the deposition of a cell wall-associated apposition called papillae for halting the fungal penetration attempt or in case of successful penetration encasements around the haustorium (40).These papillae are usually rich in callose, phenolic, and phenolic conjugates, ROS, peroxidases, cell wall structural proteins such as arabinogalactan proteins and hydroxyproline-rich glycoproteins, and cell wall polymers including pectin and xyloglucans (40). This confirms the primary role of callose in plant defense especially against biotrophs such as A. candida. In this study, callose deposition was observed through aniline blue staining under the confocal microscope. In comparison to the susceptible genotype ‘Pusa Jaikisan’, ‘Pusa Jaikisan WRR’ had higher quantum of deposition of callose. (41) also reported activation of resistance due to the deposition of callose on inner walls. This suggests a role of callose in plant defense against biotroph, however, there is still a lack of evidence to the extent of its role in determination of resistance, as it may be ascertained due to its easily detectable nature.

Apart from callose deposition, evidence till date also supports elevated levels of other defense molecules which include defense enzymes, anti-oxidative enzymes, PR proteins as well as phenolics behind an overall improved disease resistance (42). Previous studies have reported enhanced activities of these defense molecules in several crop plants such as rice, tomato and tea resistant to specific diseases (43). For this study, different biochemical parameters were taken viz., total protein, POX, CAT, SOD, PAL, PPO, total polyphenols, total proline, total chlorophyll, and total carotenoids (44).

Plants accumulate several pathological or stress related proteins either locally in the infected tissue or systemically upon the invasion by a pathogen (45). Similar findings were suggested by relationship between the cell wall-invading pathogen and protein content in plants thus defining their role in disease progression (19). However, in the present study a negative correlation was observed between protein content and disease resistance as compared to ‘Pusa Jaikisan’ the total protein was found lower in ‘Pusa Jaikisan WRR’. Similar reports have been made by many researchers. Another study evaluated the biochemical response of Brassica genotypes against A. candida and observed a negative correlation between disease and total proteins (46). The protein content was maximum in the cotyledonary leaves of susceptible cultivars.

Apart from protein, all the defense related molecules were found to be elevated in both the genotypes post-inoculation, however, the response was much more pronounced in resistant ‘Pusa Jaikisan WRR’. ROS-mediated triggering of the downstream defense signaling pathways is one of the most.

POX catalyses the oxidation of a variety of phenolic and non-phenolic electron donor substrates with H₂O₂ breakdown and is among the first enzymes to show differential response upon induction of biotic stress (47). CAT also plays an important role in converting the excess H₂O₂ produced during developmental and environmental stresses in peroxisome into water and oxygen in all aerobic species. SOD is an important antioxidant enzyme as it dismutates superoxide radicals into O₂ and H₂O₂. In this study, the resistant cultivar showed a peak in POX, CAT and SOD activity at 48 hpi followed by a gradual decline, while in case of susceptible ‘Pusa Jaikisan’ there was no significant rise in its activity. Similar results were found by(48) who noted higher POX, CAT and SOD activity in the resistant cultivar, Faisal Canola when challenged with A. candida. Other findings revealed a significant increase in the POX concentration at 48 hpi in all the cultivars upon A. candida inoculation (49). They found Donskaja, a resistant cultivar showed maximum increase however, the susceptible cultivars such as Pusa Bold and Varuna showed a minimal increment. It was also found that CAT to be higher in the resistant genotype, RH 781 as compared to the susceptible, Varuna upon A. candida inoculation (50).

PAL, another defense associated enzyme, catalyses the deamination process of phenylalanine from the primary metabolism into the crucial secondary phenylpropanoid metabolism in plants. A rise in enzymatic activity was seen at 72 hpi in the ‘Pusa Jaikisan WRR’. Previous study has elucidated the positive role of PAL in resistance against white rust as the PAL activity was higher in incompatible reactions as compared to the compatible ones (51). A negative correlation was also established between PAL concentration and disease severity in the case of Alternaria leaf blight and white rust (52). PPO catalyses the oxygen-dependent oxidation of phenols to quinone and contributes to the plant defense against plant disease and insect pests. A peculiar trend was observed in the PPO activity as it was found to be higher in ‘Pusa Jaikisan WRR’ than in ‘Pusa Jaikisan’ at 12, 24 and 48 hpi, however, a reverse trend was observed at 72 and 96 hpi. The present findings are consistent with previous studies that concluded that PPO has a positive correlation with the induction of host resistance in otherwise susceptible cultivar, EC-399301 against white rust (53). A similar trend was observed where they introduced white rust resistance genes into B. juncea cv. RL 1359 from B. napus, B. carinata and B. tournefortii (54). They observed PPO to be positively associated with the white rust-resistant trait.

Phenols are thought to play diverse functions in stressed plants, including the neutralisation of ROS, cell wall lignification, and anti-nutritional activity. The present study found total polyphenols content to be more in ‘Pusa Jaikisan WRR’ than in ‘Pusa Jaikisan’ at all the time intervals. Maximum activity was seen in ‘Pusa Jaikisan WRR’ at 72 hpi. Many researchers have reported similar trends. It was also found that total phenols and other phenolic compounds such as polyphenols have a positive role in the Indian mustard against white rust (55). Phenolic compounds and polyphenols lead to increased deposition of waxes on the leaf surface of B. juncea cultivars, and this acted as a structural barrier against the invading A. candida. It was also shown that the total polyphenols content rises with the disease progression. Donskaja had the maximum polyphenol activity at 24 hpi (56). Proline is thought to be a potent non-enzymatic antioxidant that can neutralise the detrimental effects of several ROS members. Plants accumulate high amounts of proline in response to stress (17). We recorded a higher proline content in ‘Pusa Jaikisan WRR’ as compared to the ‘Pusa Jaikisan’ at all the time intervals. There was a huge difference between both cultivars in the proline content at 0 hpi. This hints towards a possible role of proline as an in-built resistance compound. The proline content was highest at 72 hpi and 48 hpi in ‘Pusa Jaikisan’ and ‘Pusa Jaikisan WRR’ respectively. Similar reports were also made by researchers where they assessed tolerance to Alternaria blight disease by measuring the activity of oxidative enzymes in a transgenic line (BjV5) of B. juncea (57). Likewise, it was found that in the case of resistant cultivars, A. brassicae triggers proteolysis and generates cell-protecting antioxidants as seen in variety, PM-30. This results in higher proline accumulation in this genotype (58).

In the present study, total chlorophyll content was more in ‘Pusa Jaikisan WRR’ as compared to the ‘Pusa Jaikisan’ at all the time intervals. In ‘Pusa Jaikisan’, the activity saw an increment up to 48 hpi and then gradually declined. For ‘Pusa Jaikisan WRR’, the activity increased up to 48 hpi, then decreased at 72 hpi and again increased at 96 hpi. These findings are in line with former studies where a positive impact of chlorophyll was concluded on white rust resistance, and it should be a factor of consideration when screening for white rust resistance genotypes. We recorded and analysed carotenoid content in both the resistant and susceptible cultivars and found it to be higher in the former at all time intervals (59). The maximum activity in the resistant cultivar, ‘Pusa Jaikisan WRR’ was seen at 72 hpi. Another study also reported a positive relationship between total carotenoids and the disease intensity in Alternaria brassicae infected plants (60).

The above findings indicate a positive correlation between resistance and that of antioxidant enzymes (POX, CAT, and SOD), PAL, PPO, polyphenols, proline, chlorophyll, and carotenoids. The antioxidant enzymes such as POX, CAT, and SOD and the non-enzymatic ROS scavenging compounds such as polyphenols and proline increase in both resistant and susceptible cultivars, but the quantum of increase is much higher in the former. This may stem from the fact that ROS is an important determinant of cell death, and it occurs much more quickly in resistant cultivars, which ultimately necessitates faster scavenging of ROS to save the plants from excessive cell death. PAL is the first committed enzyme of the cinnamate-related secondary metabolism and is instrumental in resistance. Thus, it was found higher in the resistant cultivars. PPO was found to be increased in the resistant cultivars due to the role it plays in converting the phenols into toxic quinones which further stops the growth of the pathogen. Chlorophyll was reported to increase significantly in both the cultivars. This may be due to the induction of “green islands” by A. candida, which can fix 5 times more CO₂ than uninfected plants (61). Carotenoids were found in higher concentrations in the resistant cultivar due to the reason that they are potent ROS scavengers. A negative correlation of total protein with disease resistance was also reported in the present study. A possible reason may be that for the synthesis of most of the defense-related enzymes, the proteins are broken down thus decreasing their concentration in plants.

PR proteins plays crucial role in disease resistance along with the hormonal responses triggered by intricate signalling and networking. The PR-protein induction has been reported in various plant-fungi interactions (8). Salicylic acid (SA) and Jasmonic acid (JA) / Ethylene (ET) are two important components of hormonal signalling-mediated resistance. Both these are known to be antagonistic. In case of biotrophs, the SA-mediated resistance is activated while in the case of necrotrophic attack, an induction of the JA/ET pathway occurs. The gene-for-gene concept is positively applied to the biotrophs where the interaction between an Avr gene and an R gene results in resistance. This is further manifested by the activation of SAR and SA-dependent signalling. For this, both the concepts were integrated and PR1 and PR2 were taken as SA-marker genes and PR3 and PR12 were taken as JA/ET-marker genes to check whether both pathways are synergistic or antagonistic and which set of PR genes play a positive role in white rust resistance against B. juncea. ‘

Our findings suggest that the resistant cultivar had a higher expression of the PR1 gene at all time intervals as compared to the susceptible one. In the resistant cultivar, ‘Pusa Jaikisan WRR’, the maximum expression occurred at 48 hpi with a gradual decrease thereafter. In the susceptible cultivar ‘Pusa Jaikisan’, the expression was found to be consistent at all time intervals. Other workers observed similar findings. Similar studies also reported the PR1 protein isolated from tobacco and other solanaceous plants effectively reduced spore germination and pathogen growth in the plants (62). Overexpression of numerous PR1 genes in various plant species increased resistance to oomycetes, but the effect on other pathogens taxa is unknown. PR1 has recently been demonstrated to bind sterols, indicating a protective mode of action based on the limitation of this key nutrient for oomycetes (63, 64). In the case of PR2 gene expression, the ‘Pusa Jaikisan WRR’ showed a higher relative expression as compared to ‘Pusa Jaikisan’ at all the time intervals except at 12 hpi. Maximum expression in ‘Pusa Jaikisan WRR’ occurred at 72 hpi. No significant rise in the ‘Pusa Jaikisan’ was observed and the expression was relatively the same throughout. Several findings indicate improved resistance to oomycetes in plants due to overexpression of PR2 this could be due to the presence of β-1,3-Glucanase in cell walls of oomycetes (63).

Chitinase (PR3) catalyses the hydrolytic cleavage of chitin and is a significant antifungal enzyme. We noted a very interesting trend in PR3 gene expression where it was higher in ‘Pusa Jaikisan WRR’ at 12, 24 and 72 hpi, while it was higher at 48 and 96 hpi in ‘Pusa Jaikisan’. Maximum expression in ‘Pusa Jaikisan WRR’ occurred in the initial stages at 12 and 24 hpi with a peak at 48 hpi. At 96 hpi both cultivars showed a very similar expression. PR 12 are the most important PR proteins for necrotrophic pathogen resistance. The expression pattern of PR12 in the current study did not reveal any significant fluctuation in the ‘Pusa Jaikisan’. In ‘Pusa Jaikisan WRR’, the peak expression was seen at 48 hpi. ‘Pusa Jaikisan WRR’ had higher expression at all time points except at 12 hpi. At 96 hpi, consistent with the PR3, PR12 also had similar expression in both the cultivars. The role of PR3 and PR12 in disease resistance was elucidated by (5).They conducted a gene expression analysis that revealed upregulation of PR3 and PR12 genes only in C. sativa and S. alba as compared to B. juncea, implying their role in Alternaria resistance. This may hint towards the involvement of PR3 in general resistance mechanism against both biotrophs and necrotrophs.

The above findings indicate a positive role of PR1 and PR2 genes in the white rust resistance of B. juncea. Also, the higher expression of PR3 in the initial stages and higher expression of PR12 in the resistant cultivar may be due to the positive role of JA/ET signalling that acts complementary to the SA pathway. Due to the antagonism between SA and JA/ET signalling, JA/ET signalling is anticipated to negatively impact resistance to these pathogens. However, JA/ET signalling may also be contributory in resistance if it is active, particularly in the case of P. parasitica and Erysiphe species. The biotrophs are known to be ameliorated by SA signalling and JA/ET acts against necrotrophs and insects. These two pathways are thought to be antagonistic, but some findings suggest that JA/ET pathway is potentiating the SA pathway in resistance against the biotrophs as well. Incompatible reaction in Plasmopara viticola, a biotrophic oomycete was mediated through the JA pathway (65). This may be the case with A. candida- B. juncea interaction, where both pathways play synergistic roles.

B. juncea is one of the most important edible oilseed crops and A. candida is a serious impediment in realising its production potential. Given the pathogen’s status of being an obligate biotroph, the R-gene-based resistance is easily overcome by more virulent races. So, to develop durable resistant varieties, we need to explore the factors behind resistance. In the present study, we studied the morphological, histochemical, biochemical, and molecular parameters and their role in white rust resistance in resistant (Pusa Jaikisan WRR) and susceptible (Pusa Jaikisan) cultivars of B. juncea. Morphological studies confirmed the resistant nature of ‘Pusa Jaikisan WRR’ as there was no spot on either the adaxial or abaxial surface. Histochemical studies hinted towards a positive role of callose deposition in resistance as it was deposited in a higher quantum in ‘Pusa Jaikisan WRR’. Biochemical studies established a positive correlation between resistance and POX, CAT, SOD, PAL, PPO, polyphenols, proline, chlorophyll, and carotenoids, while there was a negative correlation between total protein and white rust resistance. In addition to these, the gene expression analysis revealed that the SA-marker genes played a crucial role in resistance, while the JA-responsive genes may also have a positive role in the white rust defense mechanism. Comparative histochemical, biochemical, and molecular studies showed that the ‘Pusa Jaikisan WRR’ activated defense response by early detection of the pathogen. Results obtained in this study will pave the way for developing strategies to induce resistance against biotrophic pathogens in susceptible cultivars by developing transgenic plants over-expressing the PR1 and PR2 genes. The role of PR3 and PR12 should also be established in the case of other biotrophic pathogens of B. juncea such as Erysiphe polygoni and Hyaloperonospora parasitica. If the role of these JA-marker genes is positive in these cases as well, then these genes can be utilised by over-expression for a broad-spectrum resistance against both biotrophic and necrotrophic pathogen complexes of B. juncea.

NIL Near isogenic lines

WRR White rust resistance

ROS Reactive oxygen species

POX Peroxidase

PAL Phenylalanine ammonia lyase

SA Salicylic acid

JA Jasmonic acid

CAT Catalase

PPO Polyphenol oxidase

Acknowledgement

The authors acknowledge Indian Council of Agricultural Research – Indian Agricultural Research Institute for providing laboratory facilities. We are grateful to ICAR-NIPB and ICAR-DRMR for their valuable insights throughout the experiment.

Author contribution

PR and LP participated in the study conception and design. PY and AS supervised the research work. PR performed laboratory work and drafted the manuscript along with figures 1 to 6 and table 1. LP, SM and DM critically reviewed the manuscript. All authors approve the final manuscript.

Funding

PR acknowledge ICAR for providing financial support through ICAR PG Fellowship.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethical approval and consent to participate

The experiment was carried out according to the relevant ethical principles and guidelines.

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

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Expression profiling, biochemical and histochemical analysis of contrasting cultivars provide insight into resistance against white rust disease (Albugo candida) in Brassica juncea L

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Abstract

Background

Results

Conclusion

Figures

Background

Methods

Plant materials and fungal materials

Inoculation test

Disease parameters

Confocal microscopy

Biochemical assays

Total Protein estimation

Peroxidase (POX) assay

Catalase (CAT) assay

Superoxide dismutase (SOD) assay

Total Polyphenols

Polyphenol oxidase (PPO) activity

Phenylalanine ammonia lyase (PAL) assay

Total proline test

Chlorophyll and carotenoid content

Gene expression profiling

Statistical analyses

Results

Phenotypic disease assessment

Comparative callose deposition

Comparative estimation of defense-related biochemical parameters

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

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