Arbuscular Mycorrhizal Fungi Trigger Transcriptional Expression of Flavonoid and Chlorogenic Acid Biosynthetic Pathways Genes in Tomato against Tomato Mosaic Virus

Tomato mosaic disease, caused by Tomato Mosaic Virus (ToMV), is one of the most destructive diseases which results in serious crop losses. Research investigations dealing with the biocontrol activity of arbuscular mycorrhizal fungi (AMF) against this viral disease are limited. In this study, the biocontrol activity of AMF on tomato plants infected with ToMV was evaluated in the greenhouse. In addition, their impacts on the transcriptional expression levels of thirteen genes controlling the phenylpropanoid, flavonoid and chlorogenic acid biosynthetic pathways were also investigated using quantitative real-time PCR. Transcriptional expressions of the majority of the studied genes were up-regulated by mycorrhizal colonization in the presence of ToMV, particularly PAL1 and HQT, suggesting their pathogen-dependent inducing effect. Under greenhouse conditions, a significant reduction in the disease severity and incidence, as well as the viral accumulation level was observed as a response to the mycorrhizal colonization of the infected plants. Moreover, the evaluated growth parameters, photosynthetic pigments, and flavonoid content were significantly enhanced by AMF colonization. The obtained results demonstrated the protective role of AMF in triggering the plant immunity against ToMV in a pathogen-dependent manner. Beside their protective and growth-promotion activities, AMF are characterized by low-cost and environment-friendly properties which support their possible use for control of tomato mosaic disease.

drought, and resistance to plant diseases 11,12 . Induction of the plant immune system using AMF against various fungal 13 , bacterial 14 , and nematode diseases 15 has been reported by many researchers during the last years, while, there are limited studies on the biocontrol activity of AMF against viral plant diseases. In this regard, Maffei et al. 16 reported a significant reduction in the disease symptoms of tomato plants infected with tomato yellow leaf curl Sardinia virus, as well as the viral DNA concentration when colonized with AMF. One of the defense-related responses triggered in the plant by AMF colonization is the accumulation of antimicrobial phenolic compounds 13 . Flavonoids and chlorogenic acid are polyphenolic compounds which have various functions in the plant including resistance against UV radiation, heat, pathogenic agents (fungi, bacteria and viruses), and herbivores, in addition to their antioxidant role 17,18 . Their antiviral properties have been reported not only against the plant viruses but also against viruses that infect human 19,20 . Their antiviral mechanisms include binding to the viral DNA and/or capsid proteins, and inhibition of the viral polymerase and integrase enzymes 21 . The present study was planned to evaluate the biocontrol potential of AMF against ToMV on tomato plants as well as their effects on the transcriptional expression levels of flavonoid and chlorogenic acid biosynthetic pathways genes.

Results
Disease assessment. Typical symptoms of tomato mosaic virus, including mosaic, mottling, yellowing, necrosis, malformation and size reduction of the plant leaves were observed on tomato plants infected with ToMV ( Fig. 1). Data presented in (Table 1) show the disease severity and incidence in response to the applied treatments. Mycorrhizal colonization of ToMV-infected plants significantly reduced both disease severity and incidence compared with the non mycorrhizal-ToMV-infected plants. No disease symptoms were observed on the uninfected plants.
Mycorrhizal colonization assessment. Levels of the mycorrhizal colonization in tomato roots of the applied treatments are presented in (Table 2). Roots of the uninfected-AMF-treated tomato plants showed high levels of the evaluated colonization parameters (colonization frequency and intensity, and arbuscules frequency), recording 88.33, 56.33, and 26.67%, respectively. Typical mycorrhizal colonization structures were observed in roots of AMF-treated tomato plants (Fig. 2). Colonization levels of the infected-AMF-treated plants showed non-significant reduction compared to the uninfected-AMF-treated plants. No mycorrhizal colonization was observed in the non-AMF-treated tomato plants.
Effects on the growth parameters. The obtained results showed that infection with ToMV significantly reduced the lengths and dry weights of tomato shoot and roots compared with the control plants, while, the number of leaves did not show any significant difference (Table 3). However, root colonization with AMF significantly enhanced all evaluated growth parameters recording the highest values when compared to the other treatments. Except for the number of leaves, AMF-colonization of the ToMV-infected plants significantly reduced the negative effects, resulted from the viral infection, on the tested growth parameters compared to the non-mycorrhizal ToMV-infected plants.  www.nature.com/scientificreports www.nature.com/scientificreports/ Effects on the photosynthetic pigments and total flavonoid content. Means of the photosynthetic pigments and the total flavonoid content of tomato plants in response to the different applied treatments are presented in (Table 4). Infection of tomato plants with ToMV led to a significant reduction in the estimated photosynthetic pigments (Chl. a and b, and carotenoids) compared to the control plants. However, colonization of tomato roots with AMF significantly enhanced all of the estimated pigments recording the highest values in this concern compared with the control plants. Mycorrhizal colonization of the ToMV-infected plants significantly enhanced the photosynthetic pigments compared with the non-mycorrhizal-infected plants. On the other hand, all applied treatments led to significant increases in the total flavonoid content of the tomato plants compared with the control plants. Total flavonoid content of the ToMV-infected plants was higher than that of the AMF-colonized plants when compared with the control plants. However, the highest total flavonoid content was noticed for the ToMV-infected-AMF-colonized plants.
transcript levels of the polyphenol biosynthesis-related genes. Expression levels of thirteen genes encoding the enzymes set catalyzing the polyphenol biosynthetic pathways were investigated 21 days after ToMV inoculation. The polyphenol biosynthetic pathway can be divided into three sections; the main phenylpropanoid biosynthetic pathway, the flavonoid biosynthetic pathway, and the chlorogenic acid biosynthetic pathway (Fig. 3).  Table 2. Colonization level of AMF in the roots of tomato plants infected with ToMV (21 days after inoculation). C = untreated control, M = colonized with AMF, and V = infected with ToMV, F = frequency of root colonization, MI = intensity of cortical colonization, and A = arbuscules frequency. *Values of each column followed by the same letter are not significantly different according to Duncan's multiple range test (P ≤ 0.05), each value represents the mean of four replicates ± SD.  www.nature.com/scientificreports www.nature.com/scientificreports/ the main phenylpropanoid biosynthetic pathway. Transcript levels of PAL1 and C4H genes, which control the first two steps in this pathway, were investigated. For PAL1 expression, treatment with either ToMV or AMF triggered the gene expression level, but the gene up-regulation in the ToMV-infected plants was much higher (60-fold) than that in the AMF-colonized plants when compared with the control plants (Fig. 4A). However, the highest PAL1 transcript level was observed for the ToMV-infected-AMF-colonized plants (116-fold). The expression level of C4H was not induced in the ToMV-infected plants, while, it decreased in the AMF-colonized plants compared with the control plants (Fig. 4A). Nevertheless, the expression level increased in the ToMV-infected-AMF-colonized plants (1.4-fold). Based on the obtained results, two points were noticed. The first, the relative expression level of PAL1 was much higher than that of C4H, suggesting that both genes are not expressed in a coordinated manner. The second, the up-regulated expression of both genes by the dual treatment (ToMV + AMF) was higher than that of the single treatments, suggesting their synergistic effect on both genes, and the pathogen-dependent inducing effect of AMF.   Table 4. Effect of root colonization with AMF on the photosynthetic pigments (mg g −1 fresh wt) and total flavonoid content (mg rutin equivalents 100 g −1 fresh wt) in the leaves of tomato plants infected with ToMV (21 days after inoculation). C = untreated control, M = colonized with AMF, and V = infected with ToMV. *Values of each column followed by the same letter are not significantly different according to Duncan's multiple range test (P ≤ 0.05), each value represents the mean of four replicates ± SD.  www.nature.com/scientificreports www.nature.com/scientificreports/ colonization up-regulated the gene expression, but the single treatment exhibited the highest expression level (2.5-fold) compared to the control plants, whilst, colonization of tomato roots with AMF did not affect the F3H expression level. Both treatments with ToMV, singly or with AMF, enhanced the transcript level of FLS1 gene. The ToMV + AMF treatment showed the highest enhancing effect on the FLS1 expression level (7.1-fold), compared to the control treatment. With regard to DFR gene, all treatments up-regulated the gene expression but the AMF treatment was more enhancer than the ToMV treatment when compared with the control treatment. The most highly expression level was noticed for the ToMV + AMF treatment, recording 71-fold increase. Viral infection with ToMV alone or in the presence of AMF increased the expression level of F3′H gene, but the single treatment (ToMV) was the most enhancing treatment, compared with the control treatment, while the mycorrhizal colonization had no effect on the transcript level of F3′H. Infection with ToMV up-regulated the expression level of AN1  the three genes indicated that the transcriptional regulation of HQT was the highest compared to the other genes. In addition, the pathogen-dependent triggering effect of AMF was also noticed for the three genes regulating the chlorogenic acid pathway.
Accumulation level of toMV. The obtained results exhibited a high accumulation level of ToMV (8.74-fold) in the ToMV-infected plants. A considerable reduction in the viral accumulation level in tomato plants treated with ToMV + AMF (2.36-fold) was observed when compared with the control plants (Fig. 5).

Discussion
Biocontrol activity of AMF has been extensively studied against various plant diseases 12,14,15,22 . Nevertheless, reports dealing with the biocontrol activity of AMF against viral plant diseases are limited. This study deals with the biocontrol activity of AMF against ToMV on tomato plants, particularly their effects on the transcriptional regulation of flavonoid and chlorogenic acid biosynthetic pathways genes.
One of the widely reported beneficial effects of mycorrhizal association is enhancing of the host plant growth 23 . In this regard, data obtained in this study showed the enhancing effect of the mycorrhizal colonization on the evaluated tomato growth parameters as well as the photosynthetic pigments content. This result is in agreement with that reported by Lin et al. 24 on Leymus chinensis plants. Mycorrhizal association improves the water and nutrients uptake of the host plant through the external mycelial network of AMF in the rhizospheric soil 25 . Production of growth hormones in mycorrhizal plants has also been described. These endogenous hormones improve the nutrients translocations, plant photosynthetic activity and metabolism 26 . This may explain the enhancing effect of AMF on the growth of tomato plants in this study. On the other hand, it is recognized that the vigorous plant is more able to resist the invading pathogens than the weak one. Moreover, plant growth enhancement by AMF plays an important role in the compensation for the disease damages.
Results obtained from the greenhouse experiment confirmed the effective biocontrol activity of root mycorrhizal colonization against infection of tomato plants with ToMV, which resulted in a considerable reduction in the viral accumulation level. This result is in line with that of Maffei et al. 16 who reported a remarkable attenuation in the disease symptoms of tomato plants infected with tomato yellow leaf curl Sardinia virus, as well as the concentration of the viral DNA, when colonized with AMF. Induction of systemic acquired resistance (SAR) in the host plant due to mycorrhizal colonization has been described by many researchers 13,27 . This resistance is mediated by jasmonate-dependant signaling pathway 28 . A number of hypersensitivity responses including physical, biochemical and molecular defense-related changes has been reported to be associated with induction of SAR such as cell wall lignification, accumulation of pathogen-toxic substances like phenolics, enzymes, and/or pathogenesis-related proteins 13 . Furthermore, up-regulation of some defense-related genes has also been reported 29 . Among the phenolic compounds induced by SAR, this study focused on the flavonoids and chlorogenic acid. Results obtained in this study revealed a pathogen-dependent inducing effect by AMF on the transcriptional levels of the majority of the studied genes. Moreover, this induction was coordinated with the highest flavonoid content in ToMV-infected-AMF-colonized plants, compared with the other treatments. This result is in agreement with that obtained by Marquez et al. 30 who reported major transcriptional up-regulations for the defense-related genes encoding simple phenols, flavonoids, and lignin in soybean plantlets infected with Fusarium virguliforme and colonized with AMF. Interestingly, the mycorrhizal + infected soybean plantlets demonstrated the largest number of up-regulated genes. Flavonoids and chlorogenic acid have various functions in the plant including resistance against biotic and abiotic stresses. Their antiviral activity has been extensively reported against various plant and human viruses 19,20 . In this regard, Krcatovic et al. 31   www.nature.com/scientificreports www.nature.com/scientificreports/ 18.5 and 31.3%, compared to 24.7% for ningnanmycin 32 . Furthermore, chlorogenic acid also showed antiviral activity against influenza A (H1N1/H3N2) virus 33 . The antiviral mechanisms utilized by flavonoids and chlorogenic acid revolve around their enzyme-inhibitory effects, particularly, against viral polymerase and integrase, in addition to their binding ability to the viral nucleic acid and/or capsid protein 21 . One of the most important observations in this study is the most highly transcriptional expression levels of PAL1 and HQT compared to the other up-regulated genes. Indeed, HQT is the primary route for the chlorogenic acid biosynthesis in the solanaceous plants. HQT catalyzes the conversion of caffeoyl CoA to chlorogenic acid, as well as the reaction which converts p-coumaroyl CoA to coumaroyl quinic acid, which will be then converted into chlorogenic acid (Fig. 3) 34 . Zhang et al. 35 reported a positive correlation between the chlorogenic acid content and the transcriptional level of HQT in Lonicera japonica. Moreover, HQT-silenced tomato plants showed 98% reduction in the chlorogenic acid content than the wild-type plants 36 . On the other hand, PAL1-encoding gene regulates the first step in the main phenylpropanoid pathway which represents the start point for the biosynthesis of many important substances such as flavonoids, coumarins, and lignans 37 . PAL1 catalyzes the conversion of phenylalanine to trans-cinnamic acid, the phenylpropanoid skeleton, which is then used in the biosynthesis of flavonoids (Fig. 3) 38 .
The C4H encoding gene, which regulates the hydroxylation of t-cinnamic acid to form p-coumaric acid, the second step in the main phenylpropanoid pathway, was also up-regulated by AMF in the presence of ToMV. HCT, C4H and C3H have a defense-related function represented in their role in the biosynthesis of the monolignols used in the cell walls lignifications 39 . HCT catalyzes the biosynthsis of two major lignin building units (guaiacyl and syringyl) 40 . HCT-silenced Nicotiana plants exhibited considerable changes in the amount and composition of lignin and affects phenylpropanoid metabolism 41 . Induction of transcriptional expression of these genes indicates their protective role against ToMV. The CHS and CHI2 encoding genes have also a protective role in the plant immunity through regulating the pathogen-dependant accumulation of flavonoids and isoflavonoid phytoalexins. Both enzymes catalyze the conversion of p-coumaroyl CoA into naringenin 42,43 . Up-regulation of CHS and CHI2 encoding genes by mycorrhizal colonization in the ToMV-infected plants enhances the plant immunity against this virus. Likewise, transcriptional regulation of FLS1, F3H and DFR encoding genes leads to accumulation of flavonols supporting plant resistance against the viral accumulation, while, F3′H is involved in the flavonols-anthocyanins biosynthesis 38 . On the contrary, the transcriptional expression levels of AN1 and AN2 encoding genes, which regulate anthocyanins biosynthesis, were down-regulated by AMF colonization. In conclusion, the present study showed the growth enhancing effect of mycorrhizal colonization with AMF on tomato plants and confirmed their role in triggering plant immunity against ToMV. Transcriptional up-regulation of most of the studied genes by AMF in a pathogen-dependent manner, particularly PAL1 and HQT, may elucidate their protective role against ToMV.

Materials and Methods
Viral inoculum and tomato cultivar. Tomato plant infected with ToMV was used to prepare the viral inoculum. Infected young leaves were grinded to collect the crude cell sap using 0.1 M phosphate buffer (pH 7). The ToMV-susceptible tomato plant (cv. Ailsa Craig) was used in this study. evaluation of AMF application under greenhouse conditions. Plastic pots (20 cm-diameter) filled with sterilized soil (clay:sand, 1:1, v/v) were used. In each pot, five healthy 28-day-old tomato seedlings were transplanted. At transplanting time, half of the used pots were inoculated with AMF inoculum (50 g pot −1 ) as a seedling bed. No fertilization was applied and the pots were regularly irrigated with tap water to near filed capacity. Three weeks after transplanting, tomato plants were mechanically inoculated with a freshly prepared ToMV inoculum. For inoculation, tomato leaves were dusted with carborundum powder (600 meshes), then the ToMV inoculum was gently rubbed onto the dusted leaves using forefinger. Inoculated plants were rinsed with tap water shortly after the inoculation. Tomato plants only treated with plain sterilized water were used as a negative control. For each treatment, five pots were used as replicates. The pots were arranged in a complete randomized design. The tested treatments in this experiment were designated as follow; control (C), virus-infected (V), colonized with AMF (M), and virus-infected and colonized with AMF (V + M). All pots were kept under greenhouse conditions at 26/20 °C day/night and 65% relative humidity.

AMF inoculum.
Disease assessment. Twenty one days after ToMV inoculation, the disease symptoms were observed in all infected pots as described by Mansour and Al-Musa 44 . Disease severity of the infected plants was scored on a six-degrees-scale based on the disease symptoms and leaf damage according to Imran et al. 45 as follows: 0 = no symptoms, 1 = 1-20%, 2 = 21-40%, 3 = 41-60%, 4 = 61-80%, and 5 = 81-100%. Values of the disease severity were then transformed to percent disease index (PDI) using the following formula: where a = number of diseased plants having the same severity grade, b = severity grade, A = total number of plants and K = highest degree of infection. The disease incidence was calculated using the following formula: www.nature.com/scientificreports www.nature.com/scientificreports/ expression analysis of polyphenol-pathway genes. Total RNA extraction and cDNA synthesis. Total RNA was extracted from the treated tomato leaves using RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer's instructions and dissolved in diethyl pyrocarbonate-treated water. The extracted RNA was then incubated with DNase for 1 h at 37 °C and quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific, USA). Reverse transcription reaction was performed in a reaction mixture (20 µL) containing 2.5 µL 10×-buffer with MgCl 2 , 2.5 µL of dNTPs (10 mM), 1 µL oligo (dT) primer (10 pmol µL −1 ), 3 µL RNA (30 ng) and 0.2 µL reverse transcriptase enzyme (M-MuLV Reverse Transcriptase, Biolabs, NewEngland) and 10.8 µL sterile water. The PCR was performed using a SureCycler 8800 thermocycler (Agilent Technologies, USA) at 42 °C for 2 h, then at 70 °C for 5 min and the cDNA was then stored at −20 °C until used.

Primer
Solyc ID Abver Sequence (5′-3′) Phenylalanine  AGT TTT TGG AAA TTG GCT TCA  GCC CCA TTC TAA GCA AGA GAA CAT C   Chalcone synthase  Solyc05g053550.2  CHSF  CHSR   CAC CGT GGA GGA GTA TCG TAA GGC  TGA TCA ACA CAG TTG GAA GGC G   Chalcone isomerase 2  Solyc02g062170  CHIF  CHIR   GGC AGG CCA TTG AAA AGT TCC  CTA ATC GTC AAT GAT CCA AGC   www.nature.com/scientificreports www.nature.com/scientificreports/ For each sample, three biological and three technical replicates were performed. The comparative C T method (2 −ΔΔCT ) was used to analyze the relative mRNA expression levels of the tested genes 50 .