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Effect of Popcorn Disease Infected Leaves on Silkworm Performance and Differential Proteome Analysis of Mulberry Popcorn Disease.

Byline: Pei-feng Yin, Xiu-xiu Li, Qi-wei Zeng, Cheng-chen Shen, Le Gao and Jian-zhang Ton

Abstract

Popcorn disease, a fungal infection is reportedly known to affect the production of mulberry, however, there is no known research data available regarding the effects of popcorn infected leaves on silkworm performance. One group of silkworms was fed infected mulberry leaves and the second silkworms group was given control mulberry leaves that were infection free. Results showed that infected leaves exhibited limited toxicity to the silkworm; however, the cocoons yield found in infected group was deeply affected. Compared with control group, the biggest body weight, cocoon weigh, cocoon shell weight, pupal weight in infected group was 9.3%, 8.8%, 11.4% and 10.2%, respectively, lower than that of control group. Although the groups obtain a similar group oviposition, group good oviposition and group oviposition rate (P>0.05), the single oviposition, single good oviposition and single oviposition rate were significantly reduced by infected leaves.

Furthermore, to elucidate the molecular resistance mechanism of fruit mulberry "Da10" i.e., popcorn disease, Two-dimensional electrophoresis (2-DE), matrix-assisted laser desorption/ ionisation time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS) and bioinformatics technique were used for characterize the differential expressed proteins. Almost 78 patho-stress responsive proteins which expression level more than 1.5-fold were identified, where 50 proteins were up-regulated and 28 proteins were down-regulated; The identified proteins were categorized into 16 classes, which are mainly including energy metabolism, gene expression regulation, oxidation-reduction reaction, cellular component and stress responses, and the stress proteins including Mn-superoxide dismutase and thaumatin-like protein.

The results means that mulberry young fruits can regulate the expression levels of multiple proteins to reply popcorn disease and these pathogenesis-related proteins provide valuable information to further study the pathogenesis of popcorn disease and disease-resistant molecular breeding in mulberry.

Key words

Mulberry sorosis, Popcorn disease, Patho-stress, Comparative proteomics, Mass spectrometry analysis.

INTRODUCTION

The history of Sericulture in China is more than 5000 years (Zhang et al., 2010 #2482), and China owns the biggest silk production in the world, which product 300000 metric tons of silkworm cocoons in 2007 (Sun et al., 2012 #2484). Regarding the mulberry leaf is the only food to silkworm, the safety of mulberry leaf is the safeguard of silkworm, however, plenty factors would affect the quantity of mulberry leaf. The pesticide is no doubt the biggest problem of mulberry leaf. Exposure of silkworm larvae to pesticides may cause acute effects on survival of the insects (Zhao et al., 2004), or sub-lethal effects on silk production and quality (Sun et al., 2012 #2484). However, limited research focus on the effect of mulberry disease on the silkworm (Gencoglan et al., 2016).

On the other side, the mulberry Popcorn disease invades in the mulberry inflorescence during flowering. This disease poses serious threats to mulberry fruit production and thus, often associated with economic losses. In order to breed disease-resistant varieties to reduce the losses linked with this disease, the understanding of mulberry response mechanism to sclerotinia at molecular level become more important. Pathogen shows variedly threats to plants. With co-evolution, different plants organs build-up different respond to invading pathogens. With the infecting, the vulnerable organs in host activate the defense system, which including enzymes activities changing and synthesize resistance-related proteins, as resistance gene (R gene), allergic reactions, signal transduction pathways and pathogenesis-related proteins (PR) to conduct biological defense (Liz et al., 2011; Gu et al., 2015).

Plenty of PRs have been recognized as the key role in the plant resistance, such as glutathione S-transferase (GST) (Overby et al., 2015), plant cell wall-degrading enzymes (PCWDEs) (Kubicek et al., 2014), chitinase and chitinase-like proteins (CHI) (Marcato et al., 2016), catalase (CAT) (Iannone et al., 2015), peroxidase (POD) (Kwon et al. 2015), superoxide dismutase (SOD) (Jing et al., 2015) and polyphenol oxidase (PPO) (Chi et al., 2014), etc. A better understanding of the pathogens induced proteins will undoubtedly contribute to its resistance mechanisms research.

Sclerotinia is dangerous in agriculture, as Arabidopsis, Tomato, kidney bean and rape are susceptible to the disease (Zhou et al., 2014; Zhao et al., 2015). In addition, this fungal diseases (usually named as popcorn disease) limited the cultivation of mulberry, which invaded in the mulberry inflorescence during flowering, thus caused a terrible taste and commercial loss of the product (Xue et al., 2014).

It is clear that, a better understanding of molecular mechanism of the mulberry response to Popcorn disease will prevent the invasion of the disease efficient. In previous proteins studies, 2-DE was utilized for mulberry study, the responsive proteins of mulberry dwarf disease was identified in mulberry leaves (Borges et al., 2015). Chen et al. (2013) optimized the methods of two-dimensional electrophoresis system of mulberry fruits, and Niu et al. (2013) separation and identification of stage-specific proteins in pistillate flowers of mulberry. A wide variety of disease resistance genes of mulberry have been isolated, including NBS, MaPGIP1, phenylalanine ammonia-lyase gene (Wang et al., 2015; Zhang, 2009). Some pathogen response proteins, such as NUDIX/mut, SOD, F-box, heat shock protein were reported also (Xianling et al., 2009).

Since limited research concern the effect of popcorn disease to the silkworm and the mechanism of the Popcorn disease infecting the mulberry. The objectives of the present research were therefore focused upon the performance of clerotium disease infected mulberry leaves on silkworm development, and the molecular mechanism of mulberry response to popcorn disease. 2-DE and mass spectrometry technology was utilized to distinguish the protein expression in the mulberry, thus revealed the disease-resistant mechanism and confirmed the resistance gene of the disease.

MATERIALS AND METHODS

Silkworms

Newly hatched silkworms species "Liang-Guang No. 2" were gathered during the year May 1st, 2015, and feed with mulberry leaves which were collected from agricultural garden of Soochow University, China. 10 days later, the silkworm was feed different mulberry leaves, and terminated at 22d.

Mulberry leaves

The pathogenic strains were popcorn disease . Leaves were selected from diseased plants which total sorosis were infected by disease last more than two years and whole plant showed a marked decline, the healthy leaves comes from the mulberry that were not infected by disease in the same environmental conditions with the same annual.

Mulberry

The healthy and diseased mulberry fruits were enclosed by transparent paper bags. After 72 h inoculation, the collected samples were rinsed for dust and dirt and sipped up surface water. The final samples were putted into 2 mL EP tubes and stored in -80AdegC liquid nitrogen, respectively. Mass spectrometric analysis of protein was carried on in Proteome analysis Center, Shanghai Institutes for Biological Sciences.

Silkworm seed production

Hatching silkworms, 1-3 instar young silkworms were feed on control and infected leaves according to the method (Greis and Petkov, 2000). The silkworms fed by infection group and control group leaves were supported by enough mulberry leaves, 3 times/d at same temperature and humidity. During the study, the effect of vitality, silkworm cocoon quality and quantity, silkworm seed quality and quantity were evaluated.

Protein extraction and two-dimensional gel electrophoresis

The frozen mulberry fruit were ground to fine powder in liquid nitrogen (Wang et al., 2008). The improved phenol extraction method was utilized for total proteins extraction, all of the reagent were precooled (-20AdegC). TCA/cetone (100 % TCA10 mL, cetone 90 mL, [beta]-mercaptoethanol 70uL) was added to frozen tissue powder (1 g), and the sample was vortexed for 5 min, then stored at -20AdegC temperature for 30 min, after centrifugation (15,000 xg at 4AdegC for 5 min), the upper phase was discarded. 0.1 M cold ammonium acetate buffer solution (ammonium acetate 0.77 g, 80% acetone 100 mL) was added to the precipitate collected, which was washed with 80% acetone two times. The proteins obtained were purified by SDS and phenol phase (1:1), and the middle phase phenol was collected after a centrifugation, the precipitated was washed two times in ammonium acetate buffer solution at -20AdegC for 8-10 h, then dissolved in lysis buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 0.5% (v/v) IPG buffer, and 1% (w/v) DTT g).

The concentration samples were detected by the Bio-Rad protein assay reagent. The qualified protein samples (800 ug) was analyzed by 2-DE. First carried isoelectric focusing on pH 3-10 IPG strip, total protein was loaded onto immobilised pH gradient (IPG) strip and rehydrated for 10-12 h. Following the procedure: 30v 12h, 500v 1 h, 1000v 1h, 8000v 8h, 500v 4h. After the strips were subjected to isoelectric focussing, the IPG strip was equilibrated in solution (6 mol/L urea, 2 mol/L thiourea, 2% CHAPS, 0.5% IPG buffer containing 1% DTT w/v for 15 min, then incubated in 4% iodoacetamide w/v for 15 min on Ettan IPGphor Isoelectric Focusing System (GE Amersham). Then IPG strip was carried the second dimension electrophoresis in SDS-PAGE gel (containing 12% polyacrylamide) for three repeats on Hofer SE 600 (GE Amersham) to separate the proteins (Parkhey et al., 2015; Shen et al., 2003).

The sample obtained was stained by silver staining for 2-DE images analysis and by CBB-R250 for mass spectrometry analysis. After decolorization, scaned by gel-specific transparency scanner (UMax Powerlook 2110XL), then analyzed by image Master 5.0 (GE Healthcare), replicates were considered to calculate volume% of all protein spots. The differential protein spots in the gel by 1.5-fold or more were selected for mass spectrometry analysis (class report ratio >=1.5).

Mass spectrometry analysis

Protein spots were decolorizated by a solution to transparent (200-400 l, 100 mmol/L NH4HCO3 /30% ACN), silver staining: 30-50uL 30mmol/L K3Fe (CN)6: 100mmol/L Na2S2O3 =1:1 (v:v). The samples were swollen at room temperature for 15 min and digested by Trypsin (Promega) at 37AdegC for 12 h. After digestion, the samples obtained was ultrasound with 100 uL 60% ACN/0.1%TFA, desalted by Ziptip (millipore).

After a redissolving in 2 uL 20% acetonitrile, the peptides were eluted onto the target plate with natural drying, added supersaturated CHCA (solvent: 50%ACN0.1%TFA) for nitrogen blasting.

The samples were identified by MALDI-TOF/TOF MS (5800 MALDI-TOF/TOF/, AB SCIEX), Test method: laser source: 355 nm Nd:YAG, accelerating voltage: 2kV, positive ions; Automatic acquisition data, sample target: 384 opti-TOF123mm x 81 mmssabsciex, matrix: CHCA, MS: 800-4000Da, selected parent ion(SNR>50) to MS/MS analysis. Two-way and gel electrophoresis system special scanner Image scanner (GE Healthcare).

The result was retrieved by NCBI database under taxonomy of Rosales and Viridiplantae with Mascot 2.2 software. Type of search were MS+MS/MS. Enzyme: Trypsin; Fixed modifications: Carbamidomethyl (C); Dynamical modifications: Oxidation (M); Mass value: Monoisotopic; Protein Mass: unrestricted; Peptide Mass Tolerance: +- 100 ppm; Fragment Mass Tolerance: +- 0.4 Da; Peptide Charge State: 1+; Max Missed Cleavages: 1. Protein score and protein CI% score over 60 and 95 respectively were successfully identified proteins, which function were annotated by online software available at http://www.geneontology.org/.

RESULTS

Effect of different leaves to the vitality of silkworm

Table I describes the vitality of silkworms (%). According to the results, the vitality of silkworms found similar in both groups, suggesting limited effects of the infected leaves over silkworms' performances.

Table I.- The effect of different leaves to the vitality of silkworm (%).

###Infection###Control###P value

###group###groups

HR of 3-instar silkworm###100.00+-0###100.00+-0###>0.05

HR of 4-instar silkworm###100.00+-0###100.00+-0###>0.05

HR of 5-instar silkworm###100.00+-0###100.00+-0###>0.05

Cocooning rate###100.00+-0###100.00+-0###>0.05

Dead silkworm cocoon rate###1.31+-2.02###1.17+-3.11###>0.05

Late pupa death rate###4.35+-2.39###4.12+-3.19###>0.05

3 instar pupation rate###97.32+-2.61 96.68+-3.04 >0.05

3 instar moth rate###92.21+-3.33 93.16+-5.42 >0.05

Effect of different leaves to the cocoon quality and quantity

Results regarding various parameters of the silkworms fed on infected and control leaves are presented (Table II). Controlled groups have more body weight, cocoon weight, Pupal weight (Pa$?0.01) versus infected group. There was no statistical difference found in cocoon shell rate (%) in both groups (Pa$?0.05).

Effect of different leaves to the silkworm seed quality and quantity

Silkworm seed quality and quantity is presented in Table III. According to the results, Spawning moth rate (%) was significantly higher in infected groups than in control group. Furthermore, group oviposition egg per 15 moths remained similar (P[greater than or equal to]0.05) in both groups. Similar trends were found in case of group good oviposition (eggs/15 moths) and group oviposition rate (%), respectively. On the other hand, single oviposition (eggs/moth), single good oviposition (eggs/moth) and single oviposition rate (%), respectively, were significantly different (Pa$?0.01) in control and infected groups. In general, control groups were found to be superior in these parameters compared with the infected groups.

Table II.- The effect of different leaves to the cocoon quality and quantity.

###Infection###Control###P value

###group###group

Body weight of###3.52+-0.09###3.88+-0.11###<0.01

silkworm (g)

Cocoon weigh (g)###1.46+-0.06###1.60+-0.04###<0.01

Cocoon shell weight (g)###0.31+-0.15###0.35+-0.01###<0.01

Cocoon shell rate (%)###21.33+-1.01###21.37+-0.39###<0.05

Pupal weight (g)###1.14+-0.03###1.27+-0.02###<0.01

Table III.- The effect of different leaves to the silkworm seed quality and quantity.

###Infection###Control###P value

###group###groups

Spawning moth rate (%)###97.13+-3.21###94.36+-2.37###0.05

(eggs/15 moths)

Group good oviposition###9 174+-357###9 424+-631###>0.05

(eggs/15 moths)

Group oviposition rate###99.69+-0.01###99.21+-1.33###>0.05

(%)

Single oviposition###627+-26###654+-17###<0.01

(eggs/moth)

Single good oviposition###625+-25###653+-17###<0.01

(eggs/moth)

Single oviposition rate###99.72+-0.03###99.87+-0.06###0.05), the single oviposition, single good oviposition and single oviposition rate were significantly reduced by infected leaves.

This indicated that due the infection, there was not enough nutrition. When the mulberry branches are infected, the moisture and nutrients in the leaves are reduced by the use of the bacteria.

Metabolism and energy production related proteins

As described above, 24.62% of aim proteins were photosynthesis related proteins. After an invasion by pathogen, the mulberry could induce 16 differentially expressed photosynthetic proteins, which means a dynamic influence of the pathogen on the host photosynthetic machinery. The up- and down-regulation of RuBisCO may be related to the complicated defense response to the pathogen.

In the present study, photosynthesis related protein Rubisco activase, gamma carbonic anhydrase, were down-regulated, where the Rubisco activase was the regulatory enzyme of Rubisco, and the gamma carbonic anhydrase (CA), a zinc metal enzyme, was the Key enzymes in carbondioxide concentrating mechanism. On the contrary, ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit was up-regulated. The down-regulated was the result of chloroplast degradation, as the up-regulation photosynthesis proteins came from ribosome fragment (Galmes et al., 2014). The carbohydrate metabolism related proteins down regulated would break the metabolic balance, cause metabolic disorder and variety of symptoms in plants (He et al., 2014).

As the young fruit exhibited a larger and redder appearance compared with normal, which can be attributed to the up-regulation of other metabolism and energy production related proteins, including: fat synthesis, fruit ripening proteins, ATP synthesis related proteins, malate dehydrogenase. It is clear that, host metabolism acceleration need more energy and substrate (Karlsson et al., 2015; Chen et al., 2008).

Protein synthesis, protein processing and amino acid metabolism related proteins were dissimilar expressed in the groups. Heat shock 70 act as protective protein under biological and abiotic stress, is a kind molecular chaperone participate in the folding and unfolding, transportation and degradation of proteins. Disulfide-isomerase (PDI) is an abundant oxidoreductase enzyme in theendoplasmic reticulum (ER).

Defense-related proteins

Some plant disease resistance related proteins were identified in the samples: S-adenosylmethionine synthase (SAMS) is closely related to transmethylation, transaminopropyl and transsulfur physiological functions, participated in the synthesis of ethylene and polyamine. In genetically modified soybean, wild soybean SAMS gene can improve its ability to resist drought, salt tolerance and low temperature (Xiao et al., 2015; Chen et al., 2014). Cysteine synthase can promote the use of sulfur, further converted into antioxidant substances of glutathione, and it's up-regulated expression can enhance the disease resistance of plants (Romero et al., 2015).

The expression of Mn-superoxide dismutase (Mn-SOD) was decreased, as Mn-SOD was regard as an important anti-oxidant enzyme, which plays a key role in resisting various stress of plants. SOD could protect cell membrane damage from oxygen or other peroxide radicals (Wang et al., 2016; Kim et al., 2014). The result means that, the defensive system of young fruits had been destroyed, which caused a lower Mn-SOD expression.

In order to resist the pathogen attacks, the host plants produce a series PR proteins, which could improve the defensive capacity of plants. Thaumatin-like protein (PR5) was identified in the sample. It can combine and degradation the components beta 1, 3 glucanase of the cell walls of fungi results from its glucanase activity. It means PR5 may contribute to against the mulberry popcorn disease pathogen (Misra et al., 2016).

Heat shock proteins play a critical role when pathogen infecting host plant (Wang et al., 2004). Hsp70 take part in plant defence responses, especially pathogen recognition, and in this study, Hsp70 were up-expressed in host. This is assumed attributed to a resistance function of the protein.

The other proteins

During pathogen-mulberry interaction, some physiologically regulated proteins were identified, such as transport, transcription factor and RNA processing related proteins. SEC13 protein was transport of proteins. NAC transcription factors play regulatory roles in diverse developmental processes and stress responses, which means, many pathways are involved resistant in host during the process.

CONCLUSIONS

The present study showed that infected leaves exhibited limited toxicity to the silkworm; however, the cocoon yields found in infected group was deeply affected. Compared with control group, the biggest body weight, cocoon weigh, cocoon shell weight, pupal weight in infected group was 9.3%, 8.8%, 11.4% and 10.2%, respectively, were lower than that of control group. As shown in Table III, although the groups obtain a similar group oviposition, group good oviposition and group oviposition rate (P>0.05), the single oviposition, single good oviposition and single oviposition rate were significantly reduced by infected leaves. Furthermore, to elucidate the molecular resistance mechanism of fruit mulberry "Da10" i.e., Popcorn disease, Two-dimensional electrophoresis (2-DE), matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS) and bioinformatics technique were used for characterize the differential expressed proteins.

Almost 78 patho-stress responsive proteins which expression level more than 1.5-fold were identified, where 50 proteins were up-regulated and 28 proteins were down-regulated; The identified proteins were categorised into 16 classes, which are mainly including energy metabolism, gene expression regulation, oxidation-reduction reaction, cellular component and stress responses, and the stress proteins including Mn-superoxide dismutase and thaumatin-like protein. The results means that mulberry young fruits can regulate the expression levels of multiple proteins to reply popcorn disease and these pathogenesis-related proteins provide valuable information to further study the pathogenesis of popcorn disease and disease-resistant molecular breeding in mulberry.

ACKNOWLEDGMENTS

This work was supported by the State Key Laboratory of Silkworm Genome Biology under Grant sklsgb2013008 from; The National Natural Science Foundation of China under Grant 31072087.

Statement of conflict of interest

Authors have declared no conflict of interest.

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Author:Yin, Pei-feng; Li, Xiu-xiu; Zeng, Qi-wei; Shen, Cheng-chen; Gao, Le; Ton, Jian-zhang
Publication:Pakistan Journal of Zoology
Article Type:Report
Date:Feb 28, 2018
Words:4989
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