Journal of Plant Physiology & Pathology ISSN: 2329-955X

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Research Article, J Plant Physiol Pathol Vol: 6 Issue: 4

A Comparative Study of Host Response against Macrophomina phaseolina Infection in Sensitive and Resistant Jute (Corchorus sp) Species Unravels a Possible Defense Mechanism

Shaheena Amin, Ahlan Ferdous, Tanima Sharker, Samira Bushra, Al-Amin, Parag Palit, Mohammad Islam and Haseena Khan*

Department of Biochemistry and Molecular Biology, Molecular Biology Lab, University of Dhaka, Dhaka, Bangladesh

*Corresponding Author : Haseena Khan
Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka, Bangladesh
E-mail: [email protected]

Received: July 23, 2018 Accepted: August 17, 2018 Published: August 22, 2018

Citation: Amin S, Ferdous A, Sharker T, Bushra S, Amin A, et al. (2018) A Comparative Study of Host Response against Macrophomina phaseolina Infection in Sensitive and Resistant Jute (Corchorus sp) Species Unravels a Possible Defense Mechanism. J Plant Physiol Pathol 6:4. doi: 10.4172/2329-955X.1000185

Abstract

Plants are inherently able to save themselves from any unfavorable conditions. However, they fail to exert ample response when encountered with pathogens like Macrophomina phaseolina which infects more than 500 crops. Wild plant species empowered by superlative mechanisms can conquest deadly pathogens, an example of which is C. trilocularis, a jute plant, resistant to this necrotrophic fungal pathogen. This study tried to ascertain the underlying resistance mechanism regarding the systemic acquired resistance (SAR) and cell wall fortification. Identification and characterization of genes involved in the systemic acquired resistant pathway implicated an activity of these genes with a basal expression in resistant species but irregular in the sensitive one (C. olitorius). Real-time expression analysis of genes involved in the synthesis of cell wall components like cellulose, hemicellulose, and lignin along with chemical and microscopic examinations signified preeminent lignification in C. trilocularis. However, this vital plant cell wall component was found to decrease in C. olitorius. The present investigation provided a deep insight into the defense strategy employed by the two species of jute at the molecular level. This study is expected to help in improving the fungal resistance of susceptible but farmer popular jute species. The same should be applicable to other vulnerable crops as well. Understanding how the pathogen is recognized, and an effective defensive response mounted, could ultimately lead to the development of novel strategies for providing durable plant resistance.

Keywords: Corchorus trilocularis; Macrophomina phaseolina; Systemic acquired resistant (SAR) pathway; Lignification; Corchorus olitorius

Introduction

Competition for survival among the living beings lies at the very basis of a defense response. Since the ‘flight or fight’ mechanism of survival is not an option for plants, they are compelled to adapt to their changing interactions for survival. Consequently, this requires an evolution of intricate mechanisms that would render them the ability to combat biotic (and abiotic) stressors. Although plant-fungal interactions are mostly friendly in nature [1], fungal pathogens are important causal agents of infectious diseases in plants. Persistent and significant crop losses have made phytopathogenic fungi a serious agronomic factor, and have thus attracted the attention of both farmers, and researchers. One such factor is Macrophomina phaseolina, a notorious fungal pathogen that possesses a large number of pathogen-host interacting genes including those for adhesion, signal transduction, cell wall breakdown, purine biosynthesis, and potent mycotoxin patulin [2]. This fungus, therefore, has the potential adaptive capacity to a range of osmotic and pH environments allowing it to infect many crop species, including major food crops (maize, sorghum), pulse crops (common bean, green gram), fiber crops (jute, cotton), and oil crops (soybean, sunflower, sesame). It is the common causative agent for diseases like charcoal rot, stem rot, and root rot which are favored by untoward conditions like high temperatures and low soil moisture. This accelerates the process of pathogenesis especially in jute [2]. Jute an annual dicotyledonous bast fiber producing plant like the flax and hemp belongs to the Malvaceae family [3]. Bangladesh a major producer of this fiber grows two commercially important species, Corchorus capsularis and Corchorus olitorius. However, Macrophomina phaseolina infection causes 30% annual yield reduction of both the jute types [2].

Corchorus trilocularis, a wild jute species, comparatively shorter in height, bushy in appearance and short-lived, has been found to be resistant to M. phaseolina in earlier studies [4-6]. This is possibly due to an efficient defense mechanism, which makes the plant impervious to fungal attack [4,7]. But the resistance mechanism and how it affects multitrophic plant-microbe interactions is largely unexplored. Plants possess many defense responses which lead to both morphologic as well as effective alterations in gene expressions at the molecular level [8]. Firstly, a pathogen has to face the cell wall, which functions as a barrier against the pathogen. Plant cell walls primarily made of cellulose and hemicellulose provide structural support followed by a secondary wall that develops inside the primary one after the cell stops growing [9]. Secondly, upon attack, various defense-related polymers are synthesized to reinforce the cell wall at the sites, where the pathogens attempt penetration [5]. Lignin, a heterogeneous polymer composed of phenolic compounds, localized and deposited in the extracellular matrix, is crucial under conditions like cell necrosis occurring from pathogen attack [10]. This confers plants’ with cell wall apposition (CWA)-mediated defense against pathogens, thus making a plant less accessible to cell wall-degrading enzymes [10].

Plants also possess different defense mechanisms involving adaptive and innate immune responses [11]. Besides, the hypersensitive response (HR), which is characterized by rapid cell death at the site of infection [11], some mechanisms of resistance which exist throughout the plant’s tissues are long-lasting, sometimes for a lifetime of the plant, and effective against a broad-spectrum of pathogens including viruses, bacteria, fungi, and oomycetes. This defense strategy has been termed systemic acquired resistance (SAR) [12]. At the molecular level, SAR is characterized by increased expression of a large number of pathogenesis-related genes (PR genes) with direct antimicrobial activity, while others work to restrict movement of the pathogen [13]. When a pathogen is detected, the plant relays a signal through a complex network of signaling molecules to transcription factors that activate SAR in both local and systemic tissues, triggered by pathogens that cause necrosis, either as part of the HR or as a symptom of the disease. An example of this is an accumulation of salicylic acid (SA), acetyl SA, or benzoic acid [13-15]. In order to assess the defense status of plants routine studies with model species, mostly imply that SAR results from the combined effects of many PR proteins rather than a specific one. Among the 17 different families of PR proteins, chitinases and β-1, 3-glucanases are found in abundance when a plant is infected by pathogens. Co-induction of these two hydrolytic enzymes exerts rapid and broad-spectrum resistance in many plant species, including pea, bean, tomato, tobacco, maize, soybean, potato, and wheat [15-17]. Besides, a significant number of defense responsive genes and miRNAs have been identified by Biswas et al in a RIL population of farmer popular jute species, C. capsularis [18].

However, the current work signified molecular identification and characterization of PR genes of the systemic acquired resistance pathway and genes involved in the synthesis of cell wall components in C. trilocularis, a naturally occurring resistant species of jute. A comparative analysis of response against M. phaseolina, carried out with a sensitive jute variety (C. olitorius) at both the seedling and mature stages found C. trilocularis to be disease free throughout the infection period. This study sheds light on the natural resistance mechanism active in jute and is expected to eventually lead to sustainable crop protection system against this deadly fungal pathogen not only for jute but also for other plants infected by this or other pathogens. It will open a realm of possibilities for developing crops resistant to M. phaseolina.

Materials and Methods

Plant material and fungal stress

Two jute species namely Corchorus olitorius variety O4 and Corchorus trilocularis were used in this study. Seeds were obtained from the collection of Molecular Biology Lab at Department of Biochemistry and Molecular Biology, in the University of Dhaka and were surface sterilized with 70% ethanol, subsequently washed in distilled water, and allowed to germinate on sterile petri dishes containing 3 mm moist filter paper (Whatman) at 30 ± 1°C in the presence of water only (for maintaining 65% relative humidity). Seeds were allowed to grow for different time intervals under the specified conditions.

M. phaseolina was cultured on PDA medium as described previously [19]. 1% hyphal suspension of Macrophomina phaseolina was sprayed on seven-days old seedlings, samples were collected at different time intervals starting from 0 to 7 days (which includes 0, 3, 5 and 7 days) for expression analysis [4]. For physiological and histochemical analyses plants were grown for 4 weeks and then plants were inoculated with the fungal hyphal suspension. Samples were collected after 2 weeks (infectious period) of growth. Two independent biological replicates were collected for each of the samples.

Gene identification by PCR with degenerate primers

Genes involved in SAR pathways like ICS, WRKY 33, glucanase, chitinase and cell wall component synthesis enzyme cellulose synthase have been identified in two species of jute. Since the jute genome data is poorly elucidated [3], degenerate primers (Table 1) for these genes were designed from gene homologs of other plant spices – Gossypium, Vitis venifera, Ricinus, Theobroma cacao and Glycine max [20]. With the help of bioinformatics tools, the primers were designed manually [21]. The degenerate primers for the desired PR genes were tested for expected product sizes (approximately 450 bp to 800 bp) by performing PCRs using genomic DNA extracted from Corchorus sp. (olitorius var O4 and trilocularis). Plant DNA was isolated using the phenol-CTAB extraction method [22].

Genes’ name Degenerate primers Annealing temperature (°C) Sequence specific primers Annealing temperature (°C) Genes’ ID Species
Isochorishmat e Synthase Forward: 5-CCWCTG ATTCGTGCYTATGG-3 54 Forward:
5-ACCCG ATTTCTGGAAGCAA-3
55 MG520205 Corchorus olitorius
Reverse:
5-CTRCTACGTGCRAGTACAACC-3
Reverse:
5-AACCGTTGCATGGGATAATG-3
MG520206 Corchorus olitorius
WRKY 33 Forward: 5-ATCCHAAGCCTCAGTCYACTAGG-3 54 Forward:
5-CAGGATCCATTGGGGAGGATG-3
57 MG520207 Corchorus olitorius
Reverse:
5-CATCATGGTTGTGCTTCCCCTC-3
Reverse:
5-CTAGGCTCTCTCACAGTTCTGC-3
MG520208 Corchorus trilocularis
Glucan endo- 1,3
-beta glucosidase
Forward:
5-CTCCCTCCHAAYAARGAAGCTGT-3
57 Forward:
5-GCTGTGGATGCTAACACACTCC-3
58 MG520209 Corchorus olitorius
Reverse:
5-AACATGGHTGHCCCTGCAACA-3
Reverse:
5-ATCAGGCTCAGATGAGTCACCC-3
MG520210 Corchorus trilocularis
Chitinase Forward:
5-GCTGCTTTCTTGGCYCAAAC-3
55 Forward:
5-GAGATTTGGATCGGACCGCGTC-3
58 MG520211 Corchorus olitorius
Reverse:
5-TGTGSWGTCATCCARAACCA-3
Reverse:
5-GTCCATACGCATGGGGATATT-3
MG520212 Corchorus trilocularis
Cellulose synthase Forward:
5-GGAAGTTGGCTGGATWTATGG-3
53 Forward:
5-TCATCACCAGCCATRGCAAGAGA-3
60 MG520213 Corchorus olitorius
Reverse:
5-CTGYTCATTYCT CCACCAGTC-3
Reverse:
5-GATGCACTGT CATGGYTGGCGA-3
MG520214 Corchorus trilocularis

Table 1: List of jute (Corchorus sp.) genes identified in this study together with their respective primers.

The presence of a specific DNA band on an agarose gel after electrophoresis was the preliminary indication of the presence of these genes in jute. For further confirmation, the bands were gel extracted using Pure Link TM Quick Gel Extraction Kit (Invitrogen) and sequenced by Sanger’s sequencing service, provided by 1st BASE, Malaysia. After obtaining the sequences, the CAP3 program [23] was used to assemble the forward and reverse sequences to produce a single contig. The assembled sequence (accession no – MG520205 to MG520214) was verified through the use of NCBI Blastn database [24] with optimization to “Somewhat Similar Sequences” against the sequence data of specific genes from plant homologs Theobroma, Ricinus, Glycin, Vitis etc.

RNA isolation and cDNA construction

Total RNA was isolated from collected seedlings at different time intervals after Macrophomina infection using the TRIzol reagent (Invitrogen, USA) by following the manufacturer’s instructions [25]. First strand cDNA synthesis was made to amplify desired genes for quantitative PCR data using oligo dT primer mix (includes: 12T, C12T, G12T, A12T) to reverse transcribe all messenger RNAs (mRNAs). 2 μg of total RNA was used to perform RT reactions with Thermo Scientific™ RevertAid™ RT Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions, modified further for jute for a reaction volume of 10 μL and carried out in a Mastercycler (Eppendorf, Germany).

Real-time expression analysis

To validate the differential expression of the desired genes at different time intervals after M. phaseolina infection in C. olitorius and C. trilocularis, real-time PCR was performed in a 32-well plate Roche LightCycler Nano System with Roche SYBR Green Master I (Roche Diagnostics, Germany) and Applied Biosystem 7500 Fast version 2.3. The real-time data analysis was completed by the log 2-ΔCq method [26] for the desired genes using gene-specific primers (Table 1). The primer sets amplified 100-200 bp within the region of interest for each gene. Tubulin was used as a reference sample for evaluating the differential expression of targeted genes at different time intervals after Macrophomina infection [21] setting the untreated samples of 0 days with respect to the fungal stress as endogenous control. All samples were amplified in technical triplicates and the mean of Cq (quantification cycle) value of each sample was used day-wise for qRT-PCR data analysis using excel data sheets [26].

Microscopic examination

To observe the physiological and anatomical changes, microscopic examinations were made with four different samples of Corchorus sp. (olitorius O4 and trilocularis) at two different time intervals, which were 4 (before stress) and 6 weeks (after stress). A free hand section was made since it is the simplest method of preparing specimens for microscopic viewing [27,28]. Two different stains were used in the study. Firstly toluidine blue (TBO); a cationic dye which binds to negatively charged groups, was used for identification of the anatomic differences amongst the species. Prepared sections were flooded (on a clean slide) with 0.1% TBO [28] for one minute followed by destaining to remove excess stain. Subsequently, one drop of 20% glycerol was added to the sections and a cover glass was applied and observed under EVOS FL imaging system for transmitant light in 10X resolution. Another dye, phloroglucinol-HCl, was used in which cinnamaldehyde end groups of lignin react to give a red-violet color [29]. A saturated solution of phloroglucinol in 20% hydrochloric acid was prepared. First phloroglucinol (about 2.0 g) was dissolved in 80 ml of 20% ethanol solution and then 20 ml of concentrated HCl (12 N) was added to it for preparing a 100 mL solution. In this study, Nikon Eclipse 50i was used for UV 2B DM 400 filter of 330 to 380 nm wavelength. Scanned images were further analyzed using ImageJ software [30] for determining the fraction of area stained with phloroglucinol (red color appearance specific for lignin).

Chemical estimation of lignin

Lignin content of the jute stem was measured to determine the amount of acid-insoluble lignin in jute for both the control and the stressed plants of C. olitorius and C. trilocularis at 5 weeks of age. To obtain the average lignin content of a plant, only the middle section (~20 cm) of the stem was used in this study. A modified Klason lignin estimation method was used to estimate acid insoluble lignin or AIL [31]. All the samples were estimated twice. The data were analyzed individually for two different sample data sets.

Data analysis

For expression profiling randomly selected seedlings were collected from treated (with M. phaseolina stress) and untreated samples from each of the two species, C. olitorius and C. trilocularis at different time intervals. For histochemical and chemical estimation of lignin, control and stressed samples of the two different plants of the same age (6 weeks) were used. Two randomly selected biological replicates and three technical triplicates for each of the samples were taken into account in expression profiling and morphological study. Standard errors were calculated by the square root of standard deviation using Microsoft excel tool pack 2013. Physiological conditions like the plants’ height and diameter were measured. For each parameter, the means, and one way ANOVA tests were performed using MS Excel.

Results

Effect of M. phaseolina on Corchorus sp.

In order to comprehend the defense status of sensitive and resistant jute species, Macrophomina phaseolina was applied to the seedlings and plants at the vegetative stage. Characteristic symptoms of M. phaseolina infection like brown lesion, loss of vigor and premature stem wilting appeared 2-3 days post infection and disease progression led to ultimate plant death after 7 days of fungal infection in the seedling stage of C. olitorius (Figure 1). In case of 6-weeks old vegetative plants, reddish-brown discoloration was followed by wilting (Figure 1). However, no such symptoms appeared either in the seedling or in the vegetative stages of C. trilocularis upon fungal inoculation throughout the observed period (Figure 1).

Figure 1: Macrophomina phaseolina application. Figure a (i-iv) M. phaseolina application to jute at the seedling stage in the two species. Figure a-i. C. olitorius after M. phaseolina infection (red box showed stressed seedlings), a-ii. C. trilocularis after M. phaseolina application (red circle, showed no disease symptom), a-iii. Control plants of C. olitorius, a-iv. Control plants of C. trilocularis. Figure b (i-iv) M. phaseolina application to vegetative stage jute plants in the two species. b-i. C. olitorius after M. phaseolina infection (red box, inset: brown lesion in the stem), b-ii. C. trilocularis after M. phaseolina application, b-iii.Control plants of C. olitorius, b-iv. Control plants of C. trilocularis.

Identification of genes involved in systemic acquired resistance (SAR) and synthesis of cell wall components in jute

The genes involved in SAR pathway namely – (i) isochorismate synthase (ICS) the rate-limiting enzyme of salicylic acid (the first signalling molecule) synthesis; (ii) WRKY group of transcription factors-which ultimately activate pathogen-related proteins, especially WRKY33 which is known to act upon fungal infestation; the PR genes mainly (iii) chitinases, (iv) glucanases were selected. Since the cell wall has an important defensive role (v) cellulose synthase gene which is involved in cellulose synthesis was also taken into account. The genes listed above were at first confirmed in jute by PCR with degenerate primers (Table 1), followed by sequencing of the respective amplicons. Sequences were confirmed and submitted to the GeneBank of NCBI database and accession numbers obtained (Table 1). The other cell wall components including: xyloglucan endo-transglycosylase/ hydrolase (XTH) involved in hemicellulose remodelling and three of the major enzymes involved in lignin synthesis viz. cinnamic 4 hydroxylases (C4H), catecholamine O methyl-transferase (COMT), ferrulate 5 hydroxylase (F5H) were confirmed by PCR with gene-specific primers [4,32].

Expression profiling of pathogenesis-related (PR) genes

Comparative expressions of the desired genes were analyzed by performing quantitative real-time PCR (qRT-PCR) between fungal infected and healthy plants of the farmer popular O-4 variety of Corchorus olitorius and Corchorus trilocularis.

In C. trilocularis an overall upregulation of the genes involved in SAR pathway (Figure 2) was observed. The ICS gene was found to be prominently expressed in the control and untreated samples although the expression decreased gradually after 7 days of stress. However, this expression was significantly higher compared to C. olitorius. In the latter, expression of ICS increased 3 days after fungal inoculation but gradually decreased until at day 7 it was almost as low as that of the control plants (Figure 2). A concomitant increase in expression of other genes involved in the SAR pathway was also observed. Transcription of WRKY 33, glucanase and chitinase (Figure 2) were high for the untreated samples of C. trilocularis, increasing further on day 3 followed by a slight decrease on day 7. This pattern was similar to that observed for ICS. In case of C. olitorius, these genes were found to express on day 3 after infection but decreased significantly up to day 7.

Figure 2: Expression of plant pathogenesis-related (PR) genes involved in systemic acquired resistant (SAR) pathway in the sensitive species, C. olitorius (denoted as O4) and resistant species C. trilocularis (denoted as Trilo) at different time intervals after Macrophomina infection.

Quantitative expression profiling of cell wall component genes

Expression of cellulose synthase and xyloglucan endo-transglycosylase were found to increase with an increase in the infection time () in the resistant species C. trilocularis. However, the result was considerably Figure 3variable in the sensitive species, C. olitorius. In the latter, the genes expressed significantly on day 3 of Macrophomina inoculation but decreased with an increase in infection time (Figure 3).

Figure 3: Expression pattern of genes of cell wall components in the sensitive species, C. olitorius (denoted as O4) and resistant species C. trilocularis (denoted as Trilo) at different time intervals after Macrophomina infection.

Expression of the genes involved in lignin synthesis such as cinnamic-4-hydroxylate (C4H), catecholamine-O-methyltransferase (COMT) and ferrulate-5-hydroxylase (F5H) have also been analyzed. They were found to be highly active before the plant encountered the pathogen and the expression increased further after M. phaseolina infection in the resistant species, C. trilocularis (Figure 3). However, the opposite was observed for C. olitorius (Figure 3), where the expression significantly increased on day 3 of fungal infection but was found to be unstable and decreased with an increase in the infection time.

Phenotypic and histochemical analysis of lignin

From quantitative real-time PCR increased expression of the lignin synthetic genes was observed in the resistant species. Therefore, further validation of lignin accumulation as one of the key adaptation tools in the resistant species was apparent. A morphological study in terms of disease symptom appearance, plant height, width, were observed in the four different 6 weeks old samples of Corchorus sp. (olitorius and trilocularis) under two different conditions; stressed (treated with M. phaseolina) and control (untreated plants). Macrophomina stress was applied to the plants and samples were collected two weeks after stress application. Characteristic symptoms of fungal infection appeared in the treated plants (Figure 1b-i) but were absent in untreated plants of C. olitorius (Figure 1b-iii). However, C. trilocularis remained unaffected (Figures 1b-ii and iv) Plant height and width were found to decrease in C. olitorius after stress application (28.60 mm and 2.17 mm respectively) compared to the control plants (31.85 mm and 3.47 mm respectively) (Table 2). But in case of the resistant species both the height and width increased in stressed plants (25.20 mm and 2.97 mm respectively) compared to the control (20.30 mm and 2.10 mm respectively) (Table 2). A lignin-specific dye phloroglucinol-HCl was used to detect lignin accumulation, in which cinnamaldehyde end groups of lignin react to give a red-violet color. Stressed and control samples of C. olitorius (var O4) and C. trilocularis were stained with phloroglucinol-HCl. Figure 4a shows a decrease in the amount of lignin in the stressed plants of O-4 (Figure 4b) compared to the control in both the hypodermis and vascular cambium. Significantly higher lignin content was observed for fungus-infected plants (Figure 4d) compared to the control (Figure 4c) of C. trilocularis. The apparent visual difference was further corroborated by ImageJ scan (Figure 4e).

Figure 4: Jute stem section stained with phloroglucinol. (a) C. olitorius var O4 control (b) C. olitorius var O4 stressed (c) C. trilocularis control (d) C. trilocularis stressed (e) Area stained in red (when stained with phloroglucinol specific for lignin) per section in control and stressed samples of C. olitorius (denoted as O4) and C. trilocularis (denoted as Trilo) after Macrophomina infection obtained from Image J software. *P<0.01 (0.009).

Species Samples Height (cm) Average height (cm) P values (Control vs. Stressed plants) Diameter (cm) Average diameter (cm) P values (Control vs. Stressed plants
C. olitorius Control 1 33.40 31.85 0.33 3.70 3.47 0.05
Control 2 30.30 3.23  
Stress 1 33.40 28.60 2.36 2.17
Stress 2 30.30 1.99  
C. trilocularis Control 1 20.80 20.30 0.03 2.69 2.41 0.25
Control 2 19.80 2.41
Stress 1 25.90 25.20 3.31 2.97
Stress 2 23.40 2.62

Table 2: Data from the physiological study of jute.

Toluidine blue was used to observe lignin localization in the control plants where anatomical differences were also visible between the sensitive and resistant species. When stained with toluidine blue, pectin appeared red or reddish purple; lignin and other phenolic compounds, green to blue-green. Thin-walled parenchyma appeared as reddish purple; collenchyma as reddish purple; lignified elements such as treachery elements and sclerenchyma appeared green to bluish-green; sieve tubes and companion cells, purple; middle lamella, red to reddish purple; callose and starch unstained (Figures 5a and b). For C. olitorius, shorter hypodermis and longer endodermis were found upon differential staining but for C. trilocularis the hypodermis was larger and the endodermis shorter.

Figure 5: Jute stem section, stained with tubulin blue. (a) C. olitorius var O4 and (b) C. trilocularis.

Lignin content

Lignin content was found to be different in the resistant and sensitive species of jute under both control and stressed conditions. In case of C. olitorius, the exact amount of lignin was found to be higher in control plants compared to the stressed ones. On the other hand, in C. trilocularis the lignin content was higher in stressed plants than in the control plants together with increased stem diameter and height (Table 2) which significantly defines lignin apposition upon M. phaseolina infection (Figure 6).

Figure 6: Percent of lignin content in control and stressed samples of C. olitorius (denoted as O4) and C. trilocularis (denoted as TRILO) after Macrophomina infection. **P<0.05 (0.035); ***P<0.05 (0.015).

Discussion

Considering the detrimental effect of Macrophomina phaseolina on yield reduction of many crop species, an effective control mechanism is an absolute necessity. This requires unveiling a plant’s inherent defense tactics. The present study was undertaken to identify the natural mechanisms active against M. phaseolina infection in plants by comparing the same between a sensitive and resistant species of jute. C. olitorius, a sensitive jute species, showed characteristic symptoms of fungal infection when fungal spore suspension was applied by spraying plants at both the seedling and vegetative stages. Reduced expression of disease-resistant genes links their roles to the progression of infection in the sensitive species. Microscopic and chemical studies likewise corroborate cell wall rupture and lignin degradation with disease development at the vegetative stage of C. olitorius (Figures 7 and 8). These implicate the pernicious effects of Macrophomina phaseolina which possesses an abundance of secreted oxidases, peroxidases, and hydrolytic enzymes for degrading cell wall polysaccharides and lignocelluloses allowing penetration of host tissue [2]. Such findings are suggestive that characteristic disease symptoms uphold the infection of M. phaseolina in C. olitorius. However, no such symptoms appeared in either the seedling or mature plants of C. trilocularis upon fungal inoculation throughout the stress period. Data regarding the expression of genes involved in different pathways along with the physiological and histological studies remarkably ascertain resistance of C. trilocularis against Macrophomina infection.

Figure 7: PCR products of PR genes in SAR pathway with degenerate primers in Corchorus olitorius var O4 (denoted as O4) and Corchorus trilocularis (denoted as Trilo). (a) Isochorishmate synthase (b) WRKY 33 (c) Glucanase (d) Chitinase. Red boxes represent the desired bands that were excised from gel and sequenced. The assembled sequences were submitted to the NCBI Database under the accession numbers MG520205 to MG520212.

Figure 8: PCR products of cell wall component genes in Corchorus olitorius var O4 (denoted as O4) and Corchorus trilocularis (denoted as Trilo). (a) Desired bands of Cellulose synthase (red box) were excised and sequenced (accession numbers MG520213 to MG520214) (Table S1) (b) Xyloglucan endo-transglycosylase; (c) C4H (d) COMT (e) F5H. Red boxes represent the desired PCR products.

In this study, PR genes such as glucanase, chitinases, and their respective upstream genes, isochorismate synthase (rate-limiting enzyme in salicylic acid) and WRKY 33 transcription factor were found to be highly expressed in C. trilocularis even before it encounters the pathogen. This persisted throughout the period during which the fungal stress was applied. However, the expression was excursive in case of the sensitive species C. olitorius. With the help of many factors including nonspecific elicitors, a basal resistance appears to establish after encountering the invading microbe [33]. This response although not effective enough to avoid total infection can induce SAR against pathogens prior to the onset of the diseased conditions [17,34]. This can be attributed to resistance in C. trilocularis that obstructs disease progression when infected with M. phaseolina. Through a sequence of events leading from an initial infection to the induction of defense genes, SAR confers quantitative long-lasting protection against a broad spectrum of microorganisms. In most cases, SAR requires salicylic acid (SA) which activates a realm of regulatory proteins that associate with the accumulation of pathogenesis-related proteins. These proteins ultimately provide defense through lignification and other structural barriers, which are contributing factors of broad-spectrum resistance [34]. An increase in expression of isochorismate synthase (ICS) upon fungal infection is expected if a plant shows response against the same, as ICS is a rate-limiting enzyme of salicylic acid synthesis pathway [35,36]. Among the different SA responsive pathways, recent studies [37-39] have shown that WRKY transcription factors especially WRKY 33, considered to respond to biotic stresses against necrotrophic fungi regulate the activities of various downstream PR proteins [40,41]. Chitinase [42,43] and β-1, 3-glucanase [44] are two important hydrolytic enzymes that are found abundantly in many plant species upon infection by different pathogens. Despite this, several studies have shown that plants transformed with both β-1, 3-glucanase and chitinase genes exhibit more resistance to certain pathogens than chitinase or β-1, 3-glucanase gene alone [42,45]. Indeed, the overall expression of genes involved in the SAR pathway was found to remain high and persistent from its initial expression up to the total infection period in the resistant species (C. trilocularis). In case of C. olitorius, gene expressions were at significant levels on day 3 of Macrophomina infection. However, these expressions did not stabilize with the progression of infection.

Again, an aroused SAR can activate further amendments of cells including cell wall fortification to counter pathogenesis, subsequently enhancing plants’ resistance to necrotrophic pathogens [42,43]. Sensing cell wall integrity is also a mechanism by which plants may detect pathogen attack [46]. In this study, expression of cellulose synthase and xyloglucan endotransglycosylase genes were found to be the same in the controls of both the species while a decrease was observed in C. olitorius upon infection. However, the expression remained the same for both the stressed and control plants of C. trilocularis. Previous physiological studies have indicated that in comparison to its high yielding counterpart C. olitorius, C. trilocularis is relatively sturdy and more adaptive [4,6]. This can be favored by an extensive xyloglucan-cellulose network which is thought to act as the principal tension-bearing structure in the cell wall [46]. Furthermore, lignin is considered a barrier against successful penetration of invasive pathogens following fortification by cellulose and hemicellulose signaling by PR genes [47]. Lignification plays a primary role in restricting pathogenesis, occurring early in the host-pathogen interaction and is localized at the site of invasion [48,49]. In this study quantitative expression analyses of genes involved in lignin biosynthesis like cinnamic 4-hydroxylase, ferrulate 5 hydroxylase and catecholamine O-methyl transferase along with chemical and microscopic measurements of lignin indicate an overall increased expression of lignin in control and stressed plants of C. trilocularis. However, in case of C. olitorius expression of the aforementioned genes and the lignin content were found to be lower compared to its control plants and the resistant species. Moreover, microscopic study of the sensitive species attests to a reduction in the lignin content leading to degradation of cell wall after Macrophomina infection (Figure 4b, yellow arrow). This is possible since the fungus possesses a wide range of lignin degrading enzymes [2]. Again, in C. olitorius, the hypodermis was found to be shorter when differentially stained with toluidine blue but for C. trilocularis it was larger (Figure 5). This can be attributed to the secondary wall thickening and lignin apposition in the hypodermis of C. trilocularis. Larger hypodermis together with lignin accumulation is prominent in adaptive plant cells occurring possibly to acquire disease resistance [50]. Consequently, the increased lignin content observed after the fungal attack in C. trilocularis (Figure 4d) apprises the prominence of lignification in resistance.

Conclusion

Due to worldwide climatic variations and environmental stressors, potential plant diseases are predicted to become more severe and widespread in the future. A better understanding of stress resistance mechanisms should shed light on management strategies for crop diseases. In this study, genes involved in the systemic acquired resistant pathway as well as cell wall fortification were found to be highly expressed in C. trilocularis, a jute species resistant to Macrophomina. On the other hand, the sensitive species C. olitorius showed aberrant expression of these genes. Again, lignification, a key defense adaptation was observed upon M. phaseolina infection in the resistant species. However, additional investigation is necessary for a thorough understanding of the interplay between regulatory sequences and their network that allow a privileged expression of defense genes in C. trilocularis. Accession numbers pertaining to the reseach are MG520205 to MG520214 in Genebank of NCBI.

References

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