The role of different innate and environmental factors in Tm-22-mediated resistance to tomato mottle mosaic virus

Tettey, Carlos Kwesi; Mu, Xiu-Qi; Ma, Hua-Yu; Chen, Xin-Yang; Geng, Chao; Tian, Yan-Ping; Yan, Zhi-Yong; Li, Xiang-Dong

doi:10.1186/s42483-023-00162-4

Research
Open access
Published: 07 March 2023

The role of different innate and environmental factors in Tm-2²-mediated resistance to tomato mottle mosaic virus

Carlos Kwesi Tettey¹,
Xiu-Qi Mu¹,
Hua-Yu Ma¹,
Xin-Yang Chen¹,
Chao Geng¹,
Yan-Ping Tian¹,
Zhi-Yong Yan ORCID: orcid.org/0000-0001-8253-0694¹ &
…
Xiang-Dong Li¹

Phytopathology Research volume 5, Article number: 8 (2023) Cite this article

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Abstract

Tomato mottle mosaic virus (ToMMV) poses a threat to production and quality of tomato fruits. The Tm-2² gene confers resistance to some tobamoviruses by recognizing viral movement proteins. However, Tm-2²-mediated resistance against ToMMV is not well known. Here, we found that ToMMV could infect wild-type but not Tm-2² transgenic Nicotiana benthamiana plants and could also infect tomato cultivar Moneymaker but not resistant cultivar Jili with homozygous Tm-2². Chimeric viral ToMMV^ToBRFV−MP with swapped ToMMV MP to MP of tomato brown rugose fruit virus could systemically infect Tm-2² transgenic N. benthamiana and tomato cultivars Jili plants. Further, transient expression of ToMMV MP in the leaves of Tm-2² transgenic N. benthamiana plants induced hypersensitive response-associated cell death, suggesting that the MP of ToMMV was the avirulent factor for the Tm-2² resistance gene. ToMMV could infect Tm-2²-containing cultivar Jinpeng 1 but not Chaobei. Sequence analysis revealed that cultivars Chaobei and Jinpeng 1 were heterozygous, where Chaobei consists of Tm-2² and Tm-2 genes, while Jinpeng 1 consists of Tm-2² and tm-2 genes. Transient co-expression assays showed that both Tm-2² and Tm-2 but not tm-2 could recognize ToMMV MP and induce hypersensitivity response-associated cell death in N. benthamiana leaves, suggesting that homozygous tomato harboring Tm-2² and heterozygous tomato containing Tm-2² and Tm-2 may exhibit more durable resistance to ToMMV than heterozygous tomato carrying Tm-2² and tm-2. Further, Tm-2² transgenic N. benthamiana and tomato cultivar Jili plants with silenced Tm-2² gene were susceptible to ToMMV. Also, silencing type-I J-domain MIP1 gene compromised Tm-2²-mediated resistance to ToMMV in Tm-2² transgenic N. benthamiana and tomato cultivar Jili. Moreover, we found that viral RNA could accumulate in the systemic leaves of Tm-2² transgenic N. benthamiana plants and tomato cultivar Jili at 35°C, but not at 20, 25, or 30°C. Altogether, our findings reveal that the Tm-2² confers resistance to ToMMV by recognizing MP, and the resistance is regulated by the allele combinations, accumulation levels of Tm-2², MIP1, and the temperature.

Background

Tomato (Solanum lycopersicum) is an economically important vegetable crop grown worldwide. However, the yield and quality of tomato fruits are seriously threatened by tobamoviruses, such as tomato mosaic virus (ToMV), tobacco mosaic virus (TMV), the emerging tomato brown rugose fruit virus (ToBRFV), and tomato mottle mosaic virus (ToMMV) (Rivarez et al. 2021). ToMMV was firstly reported in infected greenhouse tomato plants in Mexico (Li et al. 2013) and since then it has been reported in many other countries (Webster et al. 2014; Turina et al. 2016; Ambrós et al. 2017; Sui et al. 2017; Nagai et al. 2019; Lovelock et al. 2020), including China (Li et al. 2014, 2020; Chai et al. 2018; Che et al. 2018; Zhan et al. 2018; Tettey et al. 2022).

To resist viral diseases, plants have evolved resistance (R) gene-mediated innate immune systems (Kourelis and Van Der Hoorn 2018). The proteins encoded by the R genes can recognize the avirulence (Avr) factors of the viruses and trigger a cascade of signals that leads to hypersensitive response (HR) or extreme resistance at the point of infection (Dangl et al. 2013). Growing crops harboring R genes is an economically and environmentally friend way compared to the use of chemical pesticides. For this reason, many cultivated crops adopt R genes against viral infection. Examples include the N gene of Nicotiana glutinosa against TMV (Dinesh-Kumar et al. 1995; Marathe et al. 2002), L genes of pepper against some tobamoviruses (Boukema 1980), and Tm-1, Tm-2, and Tm-2² in tomato against ToMV infection (Lanfermeijer et al. 2003; Ishibashi et al. 2007).

The Tm-2, Tm-2², and tm-2 genes belong to the coiled coil-nucleotide binding site-leucine rich repeat (CC-NB-LRR) R gene family. The Tm-2 and Tm-2² alleles confer resistance to TMV and ToMV by recognizing movement proteins (MPs), whereas the tm-2 allele cannot (Weber and Pfitzner 1998; Lanfermeijer et al. 2003). The expression levels of Tm-2² influence the function of Tm-2² in resisting TMV infections (Zhang et al. 2013). Also, silencing of NbMIP1 or NbHsp90 compromised Tm-2²-mediated resistance against TMV and ToMV in the Tm-2² transgenic Nicotiana benthamiana plants (Du et al. 2013; Qian et al. 2018). However, the tm-2, Tm-2, or Tm-2² cannot mount resistance to ToBRFV (Luria et al. 2017; Hak and Spiegelman 2021; Yan et al. 2021a), and the mechanism of their resistance to ToMMV is not clear.

Temperature affects R gene-mediated resistance against some plant viruses. TMV could infect heterozygous Tm-2² tomato plants systemically at 30–31°C (Pilowsky et al. 1981). The N gene of tobacco confers resistance to TMV, but a temperature of 28°C suppressed the resistance mediated by N gene (Whitham et al. 1996). The Tsw-mediated resistance against tomato spotted wild virus (TSWV) in pepper plants is also suppressed at a continuously high temperature of 32°C (Moury et al. 1998). However, the Rx gene of potato conferred resistance to potato virus X (PVX) at a temperature of 32°C (Richard et al. 2020). Further, the R-BPMV resistance gene in Phaseolus vulgaris provides resistance against bean pod mottle virus (BPMV) at temperatures up to 35°C (Meziadi et al. 2021).

Here, we found that Tm-2² displayed resistance to ToMMV by recognizing the MP. The Tm-2²-mediated resistance was affected by Tm-2² expression levels which may be attributed to the allele combinations, MIP1 and temperature. This study will be insightful for breeding resistant tomato cultivars to ToMMV and designing cultivation management methods to control ToMMV.

Results

Tm-2 ² confers resistance to ToMMV

To determine the resistance of Tm-2² to ToMMV, we inoculated the wild-type and Tm-2² transgenic N. benthamiana plants with ToMMV. At 7 days post-agro-infiltration (dpai), the systemic leaves of the wild-type N. benthamiana plants showed mosaic and epinasty symptoms, whereas the Tm-2² transgenic N. benthamiana plants showed no symptoms (Fig. 1a). Reverse transcription-PCR (RT-PCR) analysis showed the presence of ToMMV RNA in the systemic leaves of the wild-type N. benthamiana that was agro-infiltrated with ToMMV but not in the Tm-2² transgenic N. benthamiana plants (Fig. 1b). Western blot analysis also showed a detectable level of ToMMV coat protein (CP) in the systemic leaves of the wild-type N. benthamiana plants but not the Tm-2² transgenic N. benthamiana plants (Fig. 1c). This note was also observed in tomato cultivar Jili carrying homozygous Tm-2² (Tm-2²/Tm-2²) and Moneymaker carrying homozygous tm-2 (tm-2/tm-2) plants. No symptoms were observed in ToMMV-challenged Jili tomato plants, while the tomato cultivar Moneymaker showed leaf malformation and mosaic in the systemic leaves (Fig. 1d). RT-PCR analysis also showed the presence of ToMMV RNA in the systemic leaves of the tomato cultivar Moneymaker but not in the tomato cultivar Jili (Fig. 1e). Western blot analysis also showed a detectable level of ToMMV CP in the systemic leaves of tomato cultivar Moneymaker but not Jili (Fig. 1f). These results show that Tm-2² conferred resistance to ToMMV.

The MP of ToMMV is the avirulence factor for Tm-2 ²

To determine whether the ToMMV MP gene is an Avr gene for the Tm-2² gene, we swapped the MP genes of ToMMV and ToBRFV infectious clones to generate chimeric viruses ToMMV^MP−ToBRFV and ToBRFV^MP−ToMMV, respectively (Fig. 2a). At 7 dpai, like the wild-type viruses, ToMMV^MP−ToBRFV and ToBRFV^MP−ToMMV induced epinastic viral symptoms in the wild-type N. benthamiana plants (Fig. 2b). RT-PCR detection showed the presence of viral RNA in the systemic leaves of the wild-type N. benthamiana plants (Fig. 2c). However, ToBRFV and ToMMV^MP−ToBFRV, but not ToMMV or ToBRFV^MP−ToMMV, induced epinasty symptoms on the Tm-2² transgenic N. benthamiana plants at 7 dpai (Fig. 2d). The RT-PCR detection showed the presence of viral RNA in the systemic leaves of ToBRFV and ToMMV^MP−ToBFRV inoculated Tm-2² transgenic N. benthamiana plants but not in those infiltrated with ToMMV or ToBRFV^MP−ToMMV (Fig. 2e). We also inoculated tomato cultivars Jili and Moneymaker with ToMMV, ToBRFV, ToMMV^MP−ToBFRV, and ToBRFV^MP−ToMMV. At 14 dpai, the wild-type ToBRFV and ToMMV treatments induced leaf malformation and mosaic symptoms, whereas the chimeras induced milder symptoms, including leaf mosaic and malformation symptoms in tomato cultivar Moneymaker (Fig. 2f). RT-PCR detection also showed the presence of viral RNA in systemic leaves of infected tomato cultivar Moneymaker (Fig. 2g). ToBRFV or ToMMV^MP−ToBFRV induced leaf malformation and mosaic symptoms respectively in tomato cultivar Jili, but no symptoms were observed for ToMMV or ToBRFV^MP−ToMMV (Fig. 2h). The RT-PCR detection results showed systemic infection of ToBRFV and ToMMV^MP−ToBFRV in tomato cultivar Jili (Fig. 2i).

To further confirm that the MP is an Avr factor for Tm-2², we transiently expressed the MPs of ToMMV, ToMV, TMV, and ToBRFV in Tm-2² transgenic N. benthamiana leaves. HR was induced at the point of infiltration for the MPs of ToMMV, ToMV, and TMV, but no HR was induced for ToBRFV MP (Additional file 1: Figure S1). These results show that the MP of ToMMV is the Avr factor of Tm-2².

The Tm-2 ² allele composition influenced the tomato plant resistance against ToMMV

By screening resistant tomato cultivars, we found that two cultivars, Chaobei and Jinpeng 1 which carry Tm-2², displayed distinct resistance to ToMMV. At 14 dpai, cultivar Jinpeng 1 showed leaf mosaic, stunting, and necrotic spot symptoms in systemic leaves, whereas Chaobei plants did not exhibit viral disease symptoms when challenged with ToMMV (Fig. 3a upper row). However, ToBRFV induced mosaic and deformed leaf symptoms in the systemic leaves of both Jinpeng 1 and Chaobei plants at 14 dpai (Fig. 3a lower row). RT-PCR assays using the systemic leaves showed the presence of ToMMV RNA in cultivar Jinpeng 1 but not in cultivar Chaobei at 14 dpai (Fig. 3b). However, ToBRFV RNA was detected in the systemic leaves of both Jinpeng 1 and Chaobei at 14 dpai (Fig. 3c).

To determine whether the distinct resistance observed in tomato cultivars Jinpeng 1 and Chaobei is due to the Tm2² allelic composition, we obtained the coding sequences of tm-2, Tm-2, and Tm-2² from the NCBI database and aligned them with each other. We observed that the 2135th nucleotides were C, T, and T, and 2136th nucleotides were A, C, and T, respectively, in tm-2, Tm-2, and Tm-2² (Fig. 3d). We amplified and sequenced the region spanning the 2136th nucleotide of the tm-2, Tm-2, and Tm-2² from Jinpeng 1 and Chaobei tomato cultivars. Based on the sequence chromatograms, we analyzed the allele compositions of the Tm-2 and Tm-2² in tomato cultivars Jinpeng 1 and Chaobei. The tomato cultivar Jinpeng 1 showed an overlap of nucleotides T and C at position 2135 and T and A at position 2136, signifying the presence of both Tm-2² and tm-2 alleles (TC/TA). Also, cultivar Chaobei showed no overlap with nucleotide T at position 2135 and an overlap of nucleotides T and C at position 2136, implying the presence of the Tm-2² and Tm-2 alleles (TT/TC) (Fig. 3e). The full-length sequences of Tm-2² and tm-2 from cultivar Jinpeng 1 or Tm-2² and Tm-2 from cultivar Chaobei were cloned into pCam35S binary vector to produce pCamTm-2²-Jinpeng 1, pCamtm-2-Jinpeng 1, pCamTm-2²-Chaobei, and pCamTm-2-Chaobei. The sequencing results further confirmed the allelic results along with the reference genes (Additional file 1: Figure S2). We co-expressed ToMMV MP with Tm-2²-Chaobei, Tm-2-Chaobei, Tm-2²-Jinpeng 1, and tm-2-Jinpeng 1 in wild-type N. benthamiana leaves. At 48 hpai, Tm-2-Chaobei, Tm-2²-Chaobei, and Tm-2²-Jinpeng 1 induced HR at the infiltration site, whereas tm-2-Jinpeng 1 induced no HR at the infiltration site (Fig. 3f). These results indicated that the Tm-2² allele composition might affect the resistance spectrum of tomatoes against ToMMV.

Tm-2² accumulation is important for its resistance to ToMMV

To determine whether the expression levels of Tm-2² in the transgenic Tm-2² N. benthamiana and the tomato cultivar Jili are critical for their resistance to ToMMV, we silenced Tm-2² using a TRV-based VIGS vector. At 10 dpai, RT-quantitative PCR (RT-qPCR) results showed that the silencing efficiency of Tm-2² in the Tm-2² transgenic N. benthamiana was about 85% when compared to the control plants that were inoculated with TRV-GUS (Fig. 4a). The silenced and the control Tm-2² transgenic N. benthamiana plants were then inoculated with ToMMV. At 7 dpai, viral symptoms were observed in the systemic leaves of the silenced Tm-2² transgenic N. benthamiana plants but not in the control Tm-2² transgenic N. benthamiana plants (Fig. 4b). RT-PCR results also showed that ToMMV RNA was present in the systemic leaves of the silenced Tm-2² transgenic N. benthamiana plants but not in the control plants (Fig. 4c). This experiment was repeated with the resistant tomato cultivar Jili. The silencing efficiency of Tm-2² in tomato cultivar Jili was about 90% compared to the control plants (Fig. 4d). At 14 dpai, the systemic leaves of silenced tomato cultivar Jili showed leaf necrosis, mottling, and stunting, while no symptom was observed in the control tomato cultivar Jili (Fig. 4e). RT-PCR results confirmed the presence of ToMMV RNA only in the systemic leaves of the Tm-2²-silenced tomato cultivar Jili (Fig. 4f). These results indicated that Tm-2² accumulation is important to resist ToMMV infection.

The MIP1 gene is critical for Tm-2 ²-mediated resistance against ToMMV

Silencing of the NbMIP1 is known to compromise Tm-2²-mediated resistance against TMV in Tm-2² transgenic N. benthamiana (Du et al. 2013). To determine whether S. lycopersicum MIP1 (SlMIP1) or NbMIP1 is involved in Tm-2²-mediated resistance to ToMMV, we silenced SlMIP1 and NbMIP1 using TRV-based VIGS vectors. At 10 dpai, the silencing efficiency of NbMIP1 was found to be about 95% compared to the control plants (Fig. 5a). The silenced and the control Tm-2² transgenic N. benthamiana plants were then inoculated with ToMMV. At 7 dpai, no clear symptoms were observed for the NbMIP1-silenced Tm-2² transgenic N. benthamiana plants due to distortions of the leaves (Fig. 5b). However, RT-PCR results showed the presence of viral RNA in the systemic leaves of the NbMIP1-silenced plants but not in the control plants (Fig. 5c). This result was further confirmed by Western blot analysis. The results showed ToMMV systemic infection in the NbMIP1-silenced plants but not in the control plants (Fig. 5d). We further performed the experiments in the resistant Jili tomato plants. The silencing efficiency of SlMIP1 was about 85% for Jili plants (Fig. 5e). We mechanically inoculated the control and the SlMIP1-silenced Jili plant with ToMMV. At 14 dpai, the silenced tomato plants showed epinastic and necrotic leaf symptoms, whereas the control plants showed no symptoms (Fig. 5f). RT-PCR results showed the presence of ToMMV RNA in the systemic leaves of the silenced tomato plants but not in the control plants (Fig. 5g). Western blot analysis using an anti-CP antibody showed accumulation of ToMMV CP in the systemic leaves of the SlMIP1-silenced Jili plant but not in the control plant (Fig. 5h). This result shows that the accumulation of MIP1 is also critical for Tm-2²-mediated resistance.

Temperature modulates Tm-2 ² -mediated resistance against ToMMV

To determine whether temperature variations affect Tm-2²-mediated resistance, we put ToMMV infected tomato cultivar Jili and Tm-2² transgenic N. benthamiana plants in bio-chambers at 20, 25, 30, and 35°C. At 14 dpai, no disease symptoms were observed for Jili plants under 20, 25, and 30°C conditions when compared to the control plants, revealing a decreasing disease symptom with the increasing temperatures. However, the systemic leaves (6/18 plants of cultivar Jili with three repeats) showed mild symptoms at 35°C (Fig. 6a upper panel). RT-PCR results showed that ToMMV RNA could only be detected in the systemic leaves of Jili plants exposed to 35°C (Fig. 6b). The tomato cultivar Moneymaker was infected by ToMMV under different temperatures (Fig. 6a lower panel and Fig. 6c). At 7 days post-inoculation (dpi), the systemic leaves (12/18) of Tm-2² transgenic N. benthamiana showed systemic necrosis at 35°C but not under other temperatures (Fig. 6d upper panel). The control wild-type N. benthamiana expressed variable disease symptoms under different temperatures (Fig. 6d lower panel). RT-PCR results showed the viral RNA was only present in the systemic leaves of the Tm-2² transgenic N. benthamiana plant exposed to 35°C (Fig. 6e), while viral RNA could be detected in the systemic leaves of all the wild-type N. benthamiana plants under different temperatures (Fig. 6f). These results suggest that Tm-2²-mediated resistance was compromised at 35°C, indicating that temperatures may modulate disease symptoms in susceptible plants.

Discussion

Tm-2² recognizes MP to induce resistance against ToMMV

In tomato plants, the Tm-2 and the more durable resistance gene Tm-2² were reported to confer resistance against TMV and ToMV (Lanfermeijer et al. 2003). In this study, we also observed that the Tm-2² allele was effective in controlling ToMMV infection in Tm-2² transgenic N. benthamiana plants and tomato cultivar Jili (Tm-2²/Tm-2²) (Fig. 1). These results suggest that Tm-2² gene confers resistance against ToMMV in tomato plants.

Previous reports showed that Tm-2² confers resistance to TMV and ToMV by recognizing MPs (Zhao et al. 2013; Chen et al. 2017), but Tm-2² cannot recognize ToBRFV MP to confer resistance against ToBRFV (Hak and Spiegelman 2021; Yan et al. 2021a). Here, we found that ToBRFV and ToMMV^MP−ToBRFV but not ToMMV and ToBRFV^MP−ToMMV could infect Tm-2² transgenic N. benthamiana and tomato cultivar Jili harbors Tm-2² (Fig. 2). Furthermore, transient expression of ToMMV MP induced HR-associated cell death in the leaves of Tm-2² transgenic N. benthamiana plants (Additional file 1: Figure S1). These results indicate that ToMMV MP is also the Avr factor of the Tm-2² resistance gene as that of TMV and ToMV MPs.

The allele composition and mRNA levels of Tm-2 ² define the strength of Tm-2 ²-mediated resistance to ToMMV

Tomato plants carry alleles of the resistant Tm-2, Tm-2², and non-resistant tm-2 genes (Lanfermeijer et al. 2003). The Tm-2 and Tm-2² genes are both effective in resisting most strains of ToMV by recognizing the virus MP (Weber et al. 2004). However, ToMMV can infect tomato cultivar ‘Ailsa Craig’, which harbors the Tm-2² allele (Nagai et al. 2019). Here, we found that tomato cultivars Jinpeng 1, Chaobei, and Jili contained Tm-2², and ToMMV could infect Jinpeng 1 but not Chaobei and Jili cultivars and induce disease symptoms in the plants (Figs. 1, 3). However, tomato cultivar Jinpeng 1 exhibited resistance to TMV infection (Additional file 1: Figure S3). We found that cultivar Jinpeng 1 consisted of Tm-2² and tm-2 alleles and cultivar Chaobei consisted of Tm-2² and Tm-2 alleles (Fig. 3e and Additional file 1: Figure S2), while cultivar Jili consisted of homozygous Tm-2² alleles. Similar to Tm-2², Tm-2 also recognizes ToMMV MP and induces HR-associated cell death (Fig. 3f). Previous report showed that Tm-2²-mediated extreme resistance against TMV depended on the mRNA levels of Tm-2² (Zhang et al. 2013). We also found that transgenic N. benthamiana and tomato cultivar Jili showed systemic infection of ToMMV when the Tm-2² genes were silenced (Fig. 4). The high resistance of cultivars Chaobei and Jili or susceptibility of cultivar Jinpeng 1 to ToMMV may be attributed to the different expression levels of Tm-2² and Tm-2, and other inherent factors that may also modulate Tm-2² function. Therefore, the tomato plants harboring Tm-2²/Tm-2², Tm-2²/Tm-2 alleles composition may provide more durable resistance to ToMMV than tomato plants carrying Tm-2²/tm-2. This knowledge can help tomato resistance breeding programs against ToMMV in future.

The MIP1 accumulation levels and temperature modulate Tm-2 ² -mediated resistance against ToMMV in the resistant hosts

The NbMIP1 in N. benthamiana has been reported to interact with TMV MP and Tm-2² and is required for Tm-2²-mediated resistance against TMV (Du et al. 2013). Here, we also found that silencing of the MIP1 gene in Tm-2² transgenic N. benthamiana and tomato cultivar Jili plants compromised Tm-2² resistance to ToMMV, leading to viral systemic infection (Fig. 5). This note implies that MIP1 is indispensable for Tm-2²-mediated resistance against ToMMV in N. benthamiana and tomato plants.

Temperature may also affect R gene-mediated resistance (Elad and Pertot 2014; Velásquez et al. 2018). The R-BPMV gene of Phaseolus vulgaris was effective in resisting BPMV infection up to 35°C (Meziadi et al. 2021), whereas others such as the N gene of N. tabacum lost function against TMV at 28°C (Whitham et al. 1996). TMV also can infect tomato plants harboring heterozygous Tm-2² allele under high temperatures of 30–31°C (Pilowsky et al. 1981). In our study, we observed that ToMMV infected tomato cultivar Jili (Tm-2²/Tm²-2) and Tm-2² transgenic N. benthamiana plants at 35°C but not at 30°C (Fig. 6). However, the mRNA levels of Tm-2² in Tm-2² transgenic N. benthamiana and tomato cultivar Jili at 35°C was higher than those at 30°C (Additional file 1: Figure S4), implying that the infection of plants harboring Tm-2² infected by ToMMV at 35°C is not due to a reduction in Tm-2² mRNA levels, rather the high temperature (35°C) that affects the basal factors-regulating Tm-2²-mediated resistance. Understanding these underlying mechanisms by which temperature modulates Tm-2² gene function may help in developing viral disease resistant tomato plants as well as high temperature tolerant.

Conversely, we observed that viral disease symptoms in the susceptible tomato cultivar Moneymaker and wild-type N. benthamiana were milder at 35°C than those under lower temperatures (Fig. 6), suggesting that high temperature can also attenuate symptom expression in susceptible plants. This may be due to the RNA silencing in the plants, which can be enhanced at elevated temperatures as reported earlier (Szittya et al. 2003; Velázquez et al. 2010).

Conclusions

In conclusion, we found that Tm-2² confers resistance to ToMMV and the MP of ToMMV was the Avr factor for Tm-2² and Tm-2. However, the allele composition may affect Tm-2²-mediated resistance through its accumulation levels. Also, MIP1 is required for Tm-2²-mediated resistance against ToMMV in N. benthamiana and tomato. Moreover, high temperature at 35°C could compromise Tm-2²-mediated resistance to ToMMV. This study will benefit the breeding and cultivation strategies to enhance tomato resistance against ToMMV.

Methods

Plants and viruses

The Tm-2² transgenic N. benthamiana, wild-type N. benthamiana, S. lycopersicum cultivars Jili (Shandong vegetable industry holding group, Weifang, China) with homozygosis Tm-2², Chaobei (Shandong vegetable industry holding group, Weifang, China) containing Tm-2² and Tm-2 alleles, Jinpeng 1 (Xi'an Jinpeng seed Co., Ltd., Xi'an, China) containing Tm-2² and tm-2 alleles, and Moneymaker with homozygosis tm-2 were used in this study. All the plants were grown in a greenhouse under temperature conditions of about 23°C in 16 h / 8 h (light / dark) conditions. ToMMV-SD (accession number MW373515) (Tettey et al. 2022), ToBRFV-SD isolate (accession number MK905890) (Yan et al. 2019, 2021b), and TMV-HEB2 isolate (accession number MN186255) were used in this study.

Chimeric infectious clone construction and inoculation

The MP gene sequence of ToMMV and ToBRFV was amplified and separately cloned into infectious clones pCBToBRFV and pCBToMMV to generate pCBToMMV^MP−ToBRFV and pCBToBRFV^MP−ToMMV using Ligation-Free Cloning Kit (Applied Biological Materials, Canada) following the manufacturer’s instructions. All the constructs were verified by sequencing and individually transformed into Agrobacterium tumefaciens GV3101 competent cells. The agrobacterium cultures containing the binary vectors were incubated overnight at 28°C and were shaken at 200 rpm. The pelleted cell cultures were re-suspended in MMA buffer containing 10 mM MES (pH 5.6), 10 mM MgCl_2, and 200 μM acetosyringone at OD₆₀₀ = 1.0. The fully expanded leaves of the test plants were agro-infiltrated with the cells using needleless syringes. The systemic leaves of the inoculated plants were observed for viral disease symptoms. Photographs were taken using a Canon 800D digital camera (Canon, Japan). The experiment was repeated three times using five plants for each treatment. @media print { .ms-editor-squiggler { display:none !important; } } .ms-editor-squiggler { all: initial; display: block !important; height: 0px !important; width: 0px !important; }

Determination of allele composition of the tomato cultivars

DNA was extracted from tomato cultivars using the FastPure Plant DNA Isolation kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. The region spanning the nucleotide position 2,140 was amplified using primer Tm-2²–1720-F/Tm-2²–2586-R (Additional file 2: Table S1). The allele compositions of Jinpeng 1, Chaobei, and Jili were then assessed from the DNA sequence chromatograms. The full Tm-2² allele sequences were amplified from DNA extracted from tomato cultivars Jinpeng 1 and Chaobei using primer Tm-2²–1-F and Tm-2²–2568-R (Additional file 2: Table S1) and ligated into a plasmid vector pCam::35S. The vector was transformed into Escherichia coli DH5α competent cells and the cells were plated on a selective Luria–Bertani (LB) medium containing 50 μg/ml of kanamycin, and incubated overnight at 37°C. Individual clones were selected and sequenced using the Sanger sequencing platform (Sangon Biotech, China). The sequence chromatograms were analyzed using 4Peaks software version 1.8 (Nucleobytes B.V. Gerberastraat, The Netherlands) and sequences were aligned using BioEdit version 7.2 (Informer Technologies, Inc.). The resulted sequences were aligned against reference sequences from the NCBI database (the accession numbers for tm-2, Tm-2, and Tm-2² were AF536199.1, AF536200.1, and AF536201.1, respectively). The obtained vectors containing tm-2, Tm-2, and Tm-2² were individually transformed into agrobacterium and co-expressed with ToMMV MP into leaves of wild-type N. benthamiana plants. The HR-associated cell death on the inoculated leaves was observed at 48 hpai and photographed.

Virus-induced gene silencing

About 300 bp fragments of Tm-2² (AF536201.1), NbMIP1.1a (JX271901.1) and SlMIP1 (XM_004239689.3) were amplified and individually inserted into the pTRV-2 to create pTRV2-Tm-2², pTRV2-NbMIP1, and pTRV2-SlMIP1. The pTRV2-GUS containing about 300 bp fragment of GUS was used as the control. The agrobacterium cultures containing pTRV2 derived vector were mixed with agrobacterium culture harboring pTRV1 (1:1, OD₆₀₀ = 1.0). The mixed cultures were infiltrated into 3-leaf stage Jili and Chaobei tomato plants, and 4-leaf stage Tm-2² transgenic N. benthamiana plants using a needleless syringe. The silencing efficiencies of targeted genes were determined by RT-qPCR analyses at 10 dpai. The silenced plants and the controls were mechanically inoculated with ToMMV-SD sap extract. Viral RNA accumulations in the systemic leaves of silenced plants were detected by RT-PCR and CP accumulations were detected by western blot analysis. This experiment was repeated three times using at least four plants in each repeat.

Effect of temperature on Tm-2 ²-mediated resistance

Tomato and N. benthamiana were grown at room temperature to the 2 fully expanded leaf stage and 4-leaf stage, respectively. The plants were acclimatized under temperatures of 20, 25, 30, and 35°C respectively, in plant growth chambers for 48 h and then mechanically inoculated with ToMMV sap inoculum (1:10 w/v). Disease symptoms were observed and photos were taken at 7 and 14 dpi for N. benthamiana and tomato, respectively. RNA was extracted from inoculated plants to detect the ToMMV accumulation level using RT-PCR. The experiments were repeated three times independently with at least 4 plants.

RT-qPCR analysis

Total RNA was extracted from leaves using TransZol reagent (TransGen Biotech, Beijing, China), according to the manufacturer’s protocol. DNA contamination was removed from the RNA by a 4× gDNA wiper mix (Vazyme, Nanjing, China). The complementary DNAs (cDNAs) were synthesized using HiScript II Q RT SuperMix (Vazyme, Nanjing, China) containing random primers. RT-qPCR was done using a 2× Universal SYBR Green Fast qPCR mix (Abclonal) and primers listed in Additional file 2: Table S1, to test for the expression levels of Tm-2² and MIP1 using the LC96 qPCR system (Roche, Basel, Switzerland). The expression level of the ubiquitin (UBI) gene was used to normalize the expression levels of all the genes studied.

Protein analysis

For protein assays, we extracted total proteins from systemic leaves of the test plants inoculated with ToMMV. The leaves were pulverized in liquid nitrogen using mortar and pestle and transferred into 2 mL centrifuge tubes. Extraction buffer (1:3 w/v) solution containing 100 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 0.1% NP40, 5% sucrose, and proteinase inhibitor cocktail (MedChemExpress, NJ, USA) was added. The mixture was vortexed to mix and centrifuged at 13,523 g for 10 min at 4°C. The supernatant and equal volumes of 2× sodium dodecyl sulfate (2× SDS) sample buffer were added into 1.5 mL centrifuge tubes and incubated at 95°C for 10 min. The proteins were resolved in 12% SDS-PAGE followed by western blot analysis using antibodies for CP. Chemiluminescence was observed using SuperSignal™ West Dura Luminol Enhancer (Thermo Fisher Scientific, MA, USA), and the image was captured with an SH-Focus 523 Chemiluminescence Imaging System (Shenhua Science Technology, Hangzhou, China).

Statistical analysis

The Student’s t-test and Duncan’s multiple range test were separately used for two treatments and more than two treatments to calculate the statistical difference with a confidence level of 95% (P < 0.05). All data were presented as the mean + SD. The graphs were plotted using Microsoft Excel.

Availability of data and materials

Not applicable.

Abbreviations

BPMV:: Bean pod mottle virus
CC-NB-LRR:: Coiled coil-nucleotide-binding site-leucine-rich repeat
CP:: Coat protein
dpai:: Days post-agro-infiltration
dpi:: Days post-inoculation
hpai:: Hours post-agro-infiltration
HR:: Hypersensitive reaction
MIP1:: Movement interacting protein 1
MP:: Movement protein
N. benthamiana :: Nicotiana benthamiana
PBS:: Phosphate buffer saline
RT-qPCR:: Reverse transcription-quantitative polymerase chain reaction
RT-PCR:: Reverse transcription polymerase chain reaction
TMV:: Tobacco mosaic virus
ToBRFV:: Tomato brown rugose fruit virus
ToMMV:: Tomato mottle mosaic virus
TRV:: Tobacco rattle virus
TSWV:: Tomato spotted wilt virus
UBI :: Ubiquitin
VIGS:: Virus-induced gene silencing

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Acknowledgements

We thank Professor Yule Liu of Tsinghua University for providing the seeds of Tm-2² transgenic Nicotiana benthamiana.

Funding

This study was supported by grants from the National Natural Science Foundation of China (32072387) and the ‘Taishan Scholar’ Construction Project, China (TS201712023).

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Authors and Affiliations

Shandong Province Key Laboratory for Agricultural Microbiology, Laboratory of Plant Virology, Department of Plant Pathology, College of Plant Protection, Shandong Agricultural University, Tai’an, 271018, China
Carlos Kwesi Tettey, Xiu-Qi Mu, Hua-Yu Ma, Xin-Yang Chen, Chao Geng, Yan-Ping Tian, Zhi-Yong Yan & Xiang-Dong Li

Authors

Carlos Kwesi Tettey
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Xiu-Qi Mu
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Hua-Yu Ma
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Xin-Yang Chen
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Chao Geng
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Yan-Ping Tian
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Zhi-Yong Yan
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Xiang-Dong Li
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Contributions

XL and ZY conceived the experimental idea. CKT and ZY designed and conducted the experiments. CKT, YT, and XL wrote the paper. XM, CG, HM, and XC provided technical contributions and helped to revise the paper. All the authors read and approved the final manuscript.

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Correspondence to Zhi-Yong Yan or Xiang-Dong Li.

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The authors declare that they have no competing interests.

Supplementary Information

Additional file 1.

Figure S1 Hypersensitive response-associated cell death in Tm-2² transgenic Nicotiana benthamiana leave expressing MP of ToMMV, TMV, ToMV, and ToBRFV at 48 days post-agro-infiltration. Figure S2 Alignment of Tm-2², Tm-2, and tm-2 partial nucleotide sequence from position 2132 to 2171 showing nucleotide variation at position 2134 with A, C, and T for tm-2, Tm-2, and Tm-2², respectively. Tm-2-Chaobei and Tm-2²-Chaobei indicated that the sequence is obtained from the plant of tomato cultivar Chaobei, while tm-2-Jinpeng 1 and Tm-2²-JP indicate that the sequence is obtained from tomato cultivar Jinpeng 1. Figure S3 The symptoms of tomato cultivars Jinpeng 1 and Moneymaker (MM) plants inoculated with ToMMV and TMV at 14 days post-agro-infiltration (a), and RT-PCR detection of ToMMV and TMV RNA in the systemic leaves of tomato cultivars Jinpeng 1 and Moneymaker plants (b). Figure S4 RT-qPCR analysis of relative accumulation levels of Tm-2² in the systemic leaves of tomato cultivar Jili (a) and Tm-2² transgenic Nicotiana benthamiana plants (b) at 14 days of exposure to 20, 25, 30, and 35°C. The expression levels of Tm-2² in plants exposed to 20°C were normalized to 1. The ubiquitin gene expression level served as an internal control. The values indicated the mean + SD of three biological replicates. Letters indicate the significant differences among the different treatments (Duncan multiple range tests, P < 0.05).

Additional file 2.

Table S1 The primers used in this study.

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Tettey, C.K., Mu, XQ., Ma, HY. et al. The role of different innate and environmental factors in Tm-2²-mediated resistance to tomato mottle mosaic virus. Phytopathol Res 5, 8 (2023). https://doi.org/10.1186/s42483-023-00162-4

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Received: 09 October 2022
Accepted: 20 January 2023
Published: 07 March 2023
DOI: https://doi.org/10.1186/s42483-023-00162-4

The role of different innate and environmental factors in Tm-22-mediated resistance to tomato mottle mosaic virus

Abstract

Background

Results

Tm-2 2 confers resistance to ToMMV

The MP of ToMMV is the avirulence factor for Tm-2 2

The Tm-2 2 allele composition influenced the tomato plant resistance against ToMMV

Tm-22 accumulation is important for its resistance to ToMMV

The MIP1 gene is critical for Tm-2 2-mediated resistance against ToMMV

Temperature modulates Tm-2 2 -mediated resistance against ToMMV

Discussion

Tm-22 recognizes MP to induce resistance against ToMMV

The allele composition and mRNA levels of Tm-2 2 define the strength of Tm-2 2-mediated resistance to ToMMV

The MIP1 accumulation levels and temperature modulate Tm-2 2 -mediated resistance against ToMMV in the resistant hosts

Conclusions

Methods

Plants and viruses

Chimeric infectious clone construction and inoculation

Determination of allele composition of the tomato cultivars

Virus-induced gene silencing

Effect of temperature on Tm-2 2-mediated resistance

RT-qPCR analysis

Protein analysis

Statistical analysis

Availability of data and materials

Abbreviations

References

Acknowledgements

Funding

Author information

Authors and Affiliations

Contributions

Corresponding authors

Ethics declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Supplementary Information

Additional file 1.

Additional file 2.

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About this article

Cite this article

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Keywords

Phytopathology Research

The role of different innate and environmental factors in Tm-2²-mediated resistance to tomato mottle mosaic virus

Tm-2 ² confers resistance to ToMMV

The MP of ToMMV is the avirulence factor for Tm-2 ²

The Tm-2 ² allele composition influenced the tomato plant resistance against ToMMV

Tm-2² accumulation is important for its resistance to ToMMV

The MIP1 gene is critical for Tm-2 ²-mediated resistance against ToMMV

Temperature modulates Tm-2 ² -mediated resistance against ToMMV

Tm-2² recognizes MP to induce resistance against ToMMV

The allele composition and mRNA levels of Tm-2 ² define the strength of Tm-2 ²-mediated resistance to ToMMV

The MIP1 accumulation levels and temperature modulate Tm-2 ² -mediated resistance against ToMMV in the resistant hosts

Effect of temperature on Tm-2 ²-mediated resistance