Effects of Early Infection of Tobacco Mosaic Virus on Photosynthetic Proteins and Its Control

Xiaojing XU Kunfeng SONG Fengsheng HAO Jiajia ZHAO Hongxiang GUO Weiqun LIU

Abstract[Objectives]This study was conducted to explore the optimal period of TMV control and the physiological sites that interfere with TMV infection.[Methods]Proteome analysis was performed on the host tissues （tobacco plants） at different time periods of viral infection， to verify the changes in the expression of differential protein genes and N and PR1a in the photosynthetic pathway and porphyrin metabolism and chlorophyll metabolism pathways in proteome; and the tobacco plants were treated with different preparations.[Results]The expression levels of N and PR1a in the tobacco leaves treated with preparation B reached the highest level， and the effects on the expression levels of the differential protein genes were also the most significant. The control effects of the preparations were tested by the halfleaf method， and the results showed that preparation B had a significant control effect against the early infection of the virus.[Conclusions]This study lays a foundation for the prevention and control of tobacco mosaic virus on crops.

Key wordsTobacco mosaic virus; Proteome; Photosynthetic system; Control effect

Tobacco mosaic virus （TMV） is distributed all over the world， and can infect major crops such as Solanaceae， Cucurbitaceae， Cruciferae and cause serious harm. TMV is the most invasive virus that is most harmful to crops[1]. Since the plant does not have a complete immune metabolic system like an animal， once it is infected by the virus， it will be in a state of illness for a lifetime， which makes the plant virus disease difficult to control， and also brings certain difficulties to the research and development of plant virucides. Since 1886， Mayer first discovered the tobacco mosaic virus and confirmed that the juice of the infected plant is contagious[3]. For more than 100 years， people have done a lot of research on the pathogenesis and prevention of tobacco mosaic disease， with an attempt to overcome the difficulties of virus disease prevention and control， though the effect is not obvious. Viral infection is carried out in a manner that the genome of the virus is inserted into the host genome and propagates with the hostюs raw material. In recent years， scientists have made some progress in controlling TMV infection by screening chemistry[4]and biological agents[5]， but the effect is not significant. However， when preventing and controlling the disease by bioengineering technology[6]， it is impossible to enter field production.

In order to explore the optimal period of TMV control and the physiological sites that interfere with viral infection， we collected the hostюs tissues at different time periods of viral infection， and treated them with different biological and chemical agents to monitor the effects of TMV invasion on key differential protein genes， and an experiment[7]was carried out by the half leaf method to develop agents that interfere with virus infection. This study lays a foundation for the prevention of viral diseases on crops.

Materials and Methods

Materials and reagents

Test materials： Zhongyan 100; Nicotiana tabacum cv. Xanthinc.

Trizol reagent was purchased from Invitrogen Biotechnology Co.， Ltd.; reverse transcription kit and fluorescent quantitative reagent were purchased from Vazyme Biotech Co.， Ltd.; and other conventional reagents were domestic， analytically pure.

Virus extraction， inoculation and sampling

Virus extraction： The fresh diseased leaves as the TMV source were rinsed with clean water， and absorbed with filter paper to remove residual water， followed by the removal of the main vein. According to the sampling quality， 40 times volume of phosphate buffer （0.05 mol/L， pH 7.0） was added， and after grinding， 40 times of virus juice was prepared. The juice was centrifuged， obtaining the supernatant[9]for later use.

Inoculation of the virus： The tobacco plants of Zhongyan 100 grown for about 30 d with the same growth vigor were selected， for the treatment of not spraying any preparation， spraying C （Ningnanmycin） and spraying two preparations A and B， respectively. After 24 h， the fourth leaf from the top fresh leaf was selected， and the surface was evenly sprinkled with carborundum. The leaf was inoculated for 3 times with a brush which was dipped 1 ml of the virus juice by rubbing the leaf. After 1 h of inoculation， the leaves were sprayed with clear water to facilitate the onset of the plant[10-11].

Sampling： After inoculation of the virus， samples were taken at 0， 12， 24 and 36 h， respectively. The leaf above the inoculated leaf （the third leaf） was stored at -80 →.

Analysis of gene expression

Tobacco Actin was selected as the reference gene in qRTPCR. Total RNA extraction was performed according to the Trizol kit operation manual. Realtime fluorescence quantitative PCR was performed on a Biorad IQ5 fluorescence quantitative PCR instrument. The results were analyzed by 2-┐┐CT method， wherein CT is the cycle threshold， ┐CT is the difference between the CT values of the target gene and the housekeeping gene， and ┐┐CT is the difference between the ┐CT values of the different time treatments and the control.

Verification by the half leaf method

Different preparations were applied to the left halves of the N. tabacum cv. Xanthinc leaves （10leaf stage）. After 24 h， the whole leaves were inoculated with the virus， and three leaves were inoculated for each treatment. After 5 d， the results were observed.

Results and Analysis

Differential expression of proteins in photosynthetic pathways and chlorophyll metabolism pathways at different time points after inoculation with TMV

According to previous studies， it is known that viral infection has the greatest impact on the photosynthetic system[12]， so we first analyzed the differential proteins in the photosynthetic pathway. The results showed （Fig. 1） that as the time of inoculation of TMV was prolonged， the proteins upregulated in the photosynthetic pathway gradually increased， but the types of proteins affected were different， including PSI 9kD protein， ferredoxinNADP reductase， Rieske FeS， psbPlike protein 1， and photosystem I reaction center subunit N; and the downregulated proteins were the most at 24 h. Some proteins in chlorophyll metabolism， such as magnesiumprotoporphyrin IX monomethyl ester[oxidative]cyclase， were upregulated at 12 and 24 h， and began to decrease at 36 h. From 12 to 24 h after TMV infection， some proteins in the chlorophyll metabolism pathway showed compensatory expression， to maximize the need to survive. This indicates that TMV infection affects the expression of the chloroplast genome， thereby affecting photosynthesis metabolism and the function of the internal cystidium membrane photosystems I and II.

qPCR detection of changes in differential protein gene expression

To further determine the changes of these differentially expressed proteins in the digital expression profile， realtime quantitative PCR was used to verify the gene expression changes of PsbA， PetD， PsaA， geranylegerany1 diphosphate reductase in the photosynthetic pathway and oxygenevolving enhancer protein 1 and shortchain dehydrogenase TIC 32 in porphyrin metabolism and chlorophyll metabolism pathway （Fig. 2）. The results showed that the mRNA expression levels of these differential proteins are the same as the data trends in the proteome （Table 2）.

Yuanchao XU et al. Effects of Different Extraction and Purification Methods on the Acquisition of Phycoerythrin from Porphyridium purpureum

Protection effects of different control agents on photosynthetic system and chloroplast metabolism

To validate the protective effects of the developed TMVpreventing preparations A and B on the photosynthetic system and chloroplast metabolism， we screened four proteins having significant differences after TMV infection from the proteome （Table 2） to treat tobacco leaves， with the currently commercially available ningnanmycin preparation with a better TMV control effect as the positive control. The changes of these differential protein genes were detected by qRTPCR. The results showed （Fig. 2） that the expression levels of oxygenevolving enhancer protein 1 and shortchain dehydrogenase TIC 32 in the porphyrin metabolism and chlorophyll metabolism pathway of tobacco plants treated with the B preparation were significantly higher than that of other treatment groups; and meanwhile， the decreases of PsbA， PetD， and PsaA were significantly lower than other treatment groups. The comprehensive results showed that preparation B had a significant effect on the control of TMV.

Detection of N gene and diseaserelated genes

N is a TMVresistant marker gene in tobacco[13]， and the diseaserelated gene PR1a can reflect plant resistance to disease[14]. In order to further determine the control effects of the formulated preparations on TMV， the expression levels of N and PR1a were detected after the inoculation of TMV， and compared with the control， preparation B had a better control effect （Fig. 4）.

Detection of disease spots after TMV infection

In order to further confirm the control effects of preparations A and B on TMV， the half leaf method was used for the detection （Fig. 6）， and the results showed that preparation B had a good control effect on TMV.

Conclusions and Discussion

Proteomics is currently an important technology for studying the protein composition and changes of cellular tissues and organisms. When the virus infects tobacco plants， screening out the proteins with significant differences by detecting the changes in the protein composition in the leaves of tobacco plants at different time points can allow us to understand which subcellular organs the virus infection causes damage to and their metabolic pathways， which is conducive to the prevention or alleviation of the infection process of the virus by taking effective measures.

We screened proteins with significant differences in photosynthetic pathway and porphyrin and chlorophyll metabolism pathways from the proteome at four early viral infection stages， particularly D1 （psA） protein， which is the key protein in photosystem II. These findings lay a foundation for our later research and the alleviation of viral infection.

On this basis， we also screened and sprayed two highly targeted preparations， and found that  the expression level of photosystemrelated oxygenevolving enhancer protein 1 in the tobacco plants treated with compound B was higher than that of other treatments， and the expression levels of PsbA， PetD， PsaA and geranylegerany1 diphosphate reductase decreased less than other treatments， indicating that compound B can reduce the damage of TMV to photosynthetic system; and the tobacco plants treated with compound B enhanced the expression level of geranylgeranyl diphosphate reductase of porphyrin metabolism and chlorophyll metabolism after the inoculation with TMV. The above data show that compound B can slow down the destruction of photosynthetic pathway at the early stage of TMV infection， and is conducive to the maintenance of photosynthesis of tobacco plants. Meanwhile， further detection of N and PR1a also shows that preparation B can enhance the defense system of tobacco plants and improve disease resistance.

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