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. 2023 May 16;24(9):1184–1191. doi: 10.1111/mpp.13353

Rootstock‐induced scion resistance against tobacco mosaic virus is associated with the induction of defence‐related transcripts and graft‐transmissible mRNAs

Madhu Kappagantu 1, Matthew Brandon 2, Yvette B Tamukong 2, James N Culver 1,2,✉
PMCID: PMC10423323  PMID: 37191642

Abstract

Grafting is a common horticultural practice used to confer desirable traits between rootstock and scion, including disease resistance. To investigate graft‐conferred resistance against viral diseases a novel heterografting system was developed using Nicotiana benthamiana scions grafted onto different tomato rootstocks. N. benthamiana is normally highly susceptible to tobacco mosaic virus (TMV) infection. However, specific tomato rootstock varieties were found to confer a range of resistance levels to N. benthamiana scions inoculated with TMV. Conferred resistance was associated with delays in virus accumulation and the reduction in virus spread. RNA sequencing analysis showed the enrichment of transcripts associated with disease resistance and plant stress in N. benthamiana scions grafted onto resistance‐inducing tomato rootstocks. Genome sequencing of resistance‐ and nonresistance‐conferring rootstocks was used to identify mobile tomato transcripts within N. benthamiana scions. Within resistance‐induced N. benthamiana scions, enriched mobile tomato transcripts were predominantly associated with defence, stress, and abscisic acid signalling when compared to similar scions grafted onto nonresistance‐inducing rootstock. Combining these findings suggests that graft‐induced resistance is modulated by rootstock scion transcriptional responses and rootstock‐specific mobile transcripts.

Keywords: heterograft, induced resistance, phloem mobile mRNA, systemic RNA transport, virus movement

Tomato/Nicotiana benthamiana heterograft systems displaying either susceptibility or resistance to tobacco mosaic virus were compared to identify rootstock‐induced scion responses and mobile mRNA‐associated graft‐induced resistance.

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Grafting as a horticultural practice likely dates back to the first millennium bce, where its discovery and use played a key role in the domestication of temperate fruits (Mudge et al., 2009). Currently, horticultural grafting is important in the production of fruits, ornamentals, and vegetables (Lee & Oda, 2002; Mudge et al., 2009). Grafting is often used to mitigate disease either directly through the use of resistant rootstock cultivars or indirectly through the induction of resistance in the grafted scion (Davis et al., 2008; Guan et al., 2012; Lu et al., 2020; Tsaballa et al., 2020). For example, powdery mildew‐resistant watermelon and pepper rootstock can transmit this resistance to normally susceptible scion stock, inhibiting powdery mildew leaf infections (Albert et al., 2017; Kousik et al., 2018). Similar reports of rootstock‐induced resistance have also been observed in tomato grafts against cucumber mosaic virus (CMV) and tomato spotted wilt virus (Spano et al., 2015, 2017). In another system, Rubio et al. (2013) observed that plum pox virus (PPV)‐resistant almond grafted onto stems of susceptible peach seedlings inhibited PPV infections. Thus, grafting represents a useful means to incorporate novel resistance into existing agriculturally important crops.

Unfortunately, little is known about the mechanism(s) responsible for the transmission of resistance into susceptible scion stock. A few studies have investigated specific physiological or gene expression changes that occur during graft‐induced resistance. Albert et al. (2017) noted that in a powdery mildew‐resistant pepper graft system NADPH oxidase activity, the enzyme responsible for superoxide production, was induced in grafted scions that displayed resistance. Against viral pathogens in tomato grafts displaying resistance against CMV it was found that RNAi‐associated genes including AGO2, DCL2, and RDR6 were up‐regulated in comparison to nongrafted plants (Spano et al., 2017). Grafting itself can induce general wound and defence gene activation, resulting in the activation of wound and oxidative stress‐related genes, producing levels of tolerance in response to virus infection (Spano et al., 2020). However, the role of general wound defence responses and genetic contributions of the rootstock and scion have not been well characterized.

To investigate graft‐induced resistance, we developed a heterograft system that confers rootstock‐induced scion resistance against infection by tobacco mosaic virus (TMV). The development of cross‐species or heterograft systems has been employed for the identification of phloem mobile components. In particular, graft combinations that combine transgenic and nontransgenic unions have been used to trace the systemic movement of nucleic acids and proteins (Haroldsen et al., 2012). In addition, several heterograft systems have also been developed to investigate the movement of mRNAs over long distances (Notaguchi et al., 2015; Xia et al., 2018). In this study tomato rootstock cultivars grafted to Nicotiana benthamiana were investigated for their ability to induce resistance against TMV. N. benthamiana is normally highly susceptible to TMV infection. However, we found that specific tomato rootstocks confer a range of resistance responses against TMV infections in grafted N. benthamiana scions. The development of resistance within N. benthamiana scions was associated with the induction of defence‐related genes and reduced virus infection levels. In addition, resistant scions also contained higher levels of rootstock mobile transcripts associated with stress and resistance. Thus, rootstock genotype impacts both the transcriptional response of the scion and the ability of specific mobile mRNAs to move into and accumulate within the scion.

For initial heterograft experiments the hybrid tomato rootstocks Maxifort, Dro141TX (De Ruiter Seed) and Estamino (Enza Zaden Seed) were mitre‐cut below the cotyledon leaves and grafted to similarly cut 3–5‐week‐old N. benthamiana scions (Figure S1). Grafted plants were maintained under a humidity dome for 1 week before transfer to a growth chamber at 25°C with a 12‐h light cycle. All three rootstocks by themselves show high levels of resistance against TMV‐related tomato mosaic virus (ToMV) strains 0, 1, and 2. TMV inoculations directly onto the leaves of nongrafted tomato rootstock plants also did not yield detectable systemic virus at 2 weeks postinoculation, indicating similarly high levels of resistance against TMV (data not shown).

Interestingly, the different rootstocks were observed to impact the growth of the N. benthamiana scion (Figure S1). Specifically, Maxifort and Estamino rootstocks resulted in larger leaves with lengths averaging 7–8 cm, while the average leaf length for Dro141TX was 5 cm (Figure S1). Infectivity assays were done at 2–3 weeks postgrafting with the first two to four expanded N. benthamiana leaves above the graft junction dusted with carborundum and mechanically inoculated with 0.2 μg/mL of purified TMV or TMV tagged with green fluoresent protein (TMV‐GFP) (Shivprasad et al., 1999). TMV infection studies showed systemic symptom development between 7 and 14 days postinoculation (dpi) in Maxifort‐ and Estamino‐grafted N. benthamiana. Most noticeable was the development of vascular necrosis within the Maxifort‐ and Estamino‐grafted N. benthamiana scion, causing the upper stem portions of the plants to collapse in a manner that was identical to what is observed in stems of nongrafted N. benthamiana similarly inoculated with TMV (Figure 1a,c). In comparison Dro141TX‐grafted N. benthamiana displayed some leaf necrosis on the inoculated leaves but no significant systemic symptoms (Figure 1b). Infection levels as measured by western immunoblot for the detection of TMV coat protein within the inoculated leaves revealed significantly higher levels of TMV in Maxifort‐ and Estamino‐ as compared to Dro141TX‐grafted N. benthamiana scions at 4 and 6 dpi (Figures 1d and S5) (Dardick et al., 2000; Padmanabhan et al., 2008; Schneider et al., 2012).

FIGURE 1.

FIGURE 1

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Susceptibility differences within tomato‐grafted Nicotiana benthamiana scions. (a–c) Tobacco mosaic virus (TMV)‐inoculated heterografts at 2 weeks postinoculation. Red arrows point to regions of virus‐induced vascular collapse. (d) TMV coat protein levels measured by western immunoblot of tissue samples taken from eight inoculated N. benthamiana scion leaves (Figure S4). Each bar represents the average level of TMV coat protein ± standard deviation. For each time point bars connected by the same letter are not significantly different (t test, α = 0.05). dpi, days postinoculation.

To further assess the level of resistance conferred within this heterograft system, subsequent studies focused on comparisons between the resistance‐conferring rootstock Dro141TX and the nonresistance‐conferring rootstock Maxifort infected with TMV‐GFP (Shivprasad et al., 1999). The number of TMV‐GFP infection foci on inoculated N. benthamiana scion leaves was lower both on average and range in Dro141TX‐grafted plants (Figure 2a,b,d). Furthermore, TMV‐GFP fluorescent infection sites were smaller and spread more slowly and showed less fluorescence over time on Dro141TX‐grafted plants than on Maxifort‐grafted scions. At 10 dpi TMV‐GFP was observed to rapidly spread into systemic leaves of Maxifort grafts but not into those of Dro141TX (Figure 2c). To monitor virus replication the relative levels of (+) and (−) strand viral RNA were measured via reverse transcription‐quantitative PCR (RT‐qPCR) for TMV‐GFP infection foci at 8 dpi (Figure 2e,f and Table S1). The results indicated no significant difference between Dro141TX‐ and Maxifort‐grafted N. benthamiana infection foci centres or edges for either (+) or (−) strand genomes. Resistance conferred by Dro141TX is primarily the combined result of reduced virus infection and spread, both within the inoculated and systemic tissues.

FIGURE 2.

FIGURE 2

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Infection of tobacco mosaic virus tagged with green fluorescent protein (TMV‐GFP) within tomato rootstock grafted Nicotiana benthamiana scions. (a–c) TMV‐GFP inoculated and systemic N. benthamiana scion leaves. (d) Number of TMV‐GFP infection foci per equivalent inoculated leaf area. Boxes show the range of foci numbers within a 9.6 cm2 area from at least 10 leaves on five different grafted plants at 8 days postinoculation. Boxes with different letters are significantly different (Wilcoxon test, α = 0.01). (e, f) Reverse transcription‐quantitative PCR analysis for + and − strand TMV‐GFP RNA within the centre and edge of individual infection foci. Bars represent the average of six to nine infection foci ± standard error and those connected by the same letter are not significantly different (t test, α = 0.05). dpi, days postinoculation; Dro, Dro141TX rootstock; Max, Maxifort rootstock.

To investigate the effect of rootstock genotype on scion gene expression, RNA sequencing (RNA‐Seq) analysis was performed on three noninfected biological replicates of N. benthamiana scions grafted onto Dro141TX or Maxifort tomato rootstock (GEO accession: GSE211493). Each cDNA library generated an average of 100 million 150‐bp paired‐end sequence reads. Over 78% (Dro141TX rootstock) and 86% (Maxifort rootstock) of these reads mapped to the N. benthamiana genome and pairwise comparisons showed that the three biological replicates from each rootstock had a high degree of similarity within the graft treatments (Figure S2). Two‐way comparison of scion transcripts between Dro141TX‐ and Maxifort‐grafted N. benthamiana tissue identified 6745 genes that displayed two‐fold (false discovery rate < 0.05) or greater differential expression with 4209 of these genes up‐regulated in Dro141TX‐grafted tissue and 2536 up‐regulated in Maxifort‐grafted scions (Table S2). A BLAST analysis was used to identify the best Arabidopsis gene match for the differentially expressed N. benthamiana genes. Gene ontology (GO) analysis was done for genes that displayed a two‐fold alteration on Dro141TX rootstocks as compared to Maxifort rootstock (Figure 3). Among up‐regulated genes on the Dro141TX rootstock we identified several categories associated with defence and stress. For example, 28 resistance genes, several of which are known to target pathogen‐associated molecular pattern (PAMP)‐ or effector‐triggered immunities (ETI) against bacterial, oomycete, and viral pathogens, were identified as up‐regulated in Dro141TX‐grafted scions (Table S2) (Kim et al., 2009; Mohr et al., 2010; Takahashi et al., 2012). Additionally, both EDS1 and PAD4 genes that encode nucleocytoplasmic lipase‐like proteins known to be controllers of basal immunity and ETI also displayed up‐regulation in Dro141TX‐grafted scions (Dongus & Parker, 2021). Also up‐regulated was ADR1, which in combination with EDS1‐PAD4 is known to promote basal immunity (Bhandari et al., 2019; Dongus & Parker, 2021). A key outcome of the EDS1‐PAD4‐ADR1 interaction is the activation of salicylic acid (SA)‐dependent pathways (Castel et al., 2019; Cui et al., 2018). Promotion of SA defence pathways occurs by the reduction of jasmonic acid (JA) directed SA‐antagonism through the inhibition of MYC2, a key regulator of JA. Interestingly, MYC2 was up‐regulated in Dro141TX‐grafted scions as well as several JA‐associated defence responses (Table S2). Thus, both SA and JA pathways directing basal and ETI‐associated resistance are activated in the Dro141TX–N. benthamiana graft combination.

FIGURE 3.

FIGURE 3

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Gene ontology analysis of Nicotiana benthamiana scion transcriptional changes. RNA‐Seq analysis derived from three biological replicates were analysed using the CLC Genomic Workbench v. 10.0.1. Reads were mapped to the N. benthamiana genome (Niben genome v1.0.1) and used to perform RNA‐Seq and differential expression analysis. A local MultiBlast search for Niben scaffolds was performed using Arabidopsis annotated genome (TAIR10). Gene ontology analysis was conducted using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) tool (Benjamini correction <0.05) (Sherman et al., 2022).

Interestingly, genes involved in phosphoinositide dephosphorylation represented the most significantly enriched GO term for Dro141TX‐grafted scions. Phosphatidylinositols are membrane glycerophospholipids that function as signalling molecules in several stress and developmental responses including SA‐associated defence (Antignani et al., 2015). Regulation of these responses is controlled through the phosphorylation status of specific phosphatidylinositol sites that occur in response to PAMP‐ and ETI‐based resistance (Zhang & Xiao, 2015). Specific responses associated with this phosphorylation include the induction of reactive oxygen species, hypersensitive defence, and stomatal closing during pathogen infection, all GO features specifically enriched in Dro141TX‐grafted scions (Zhang et al., 2020).

The most common and significantly enriched gene functions in Maxifort‐grafted scions involved genes associated with cell replication and growth, which corresponds with the enhanced leaf growth observed in N. benthamiana scions (Figures 3 and S1). These include various genes involved in DNA replication, mitotic cell division, and cell cycle control (Table S2). Additional genes are involved in ribosome biogenesis and cell wall expansion and growth. Many of these overexpressed regulatory molecules have been linked to root, cell wall, and vascular growth and development (Fisher & Turner, 2007; Shinohara et al., 2016; Wang et al., 2013). Other genes linked to plant growth that show increased expression in Maxifort‐grafted scions include six minichromosome maintenance genes that confer DNA helicase activity. These genes are involved in DNA replication and highly expressed in newly developing and proliferating tissues (Tuteja et al., 2011). Furthermore, knocking out MCM4 in Arabidopsis significantly decreases plant size (Gonzalez et al., 2020).

Approximately 12%–20% of RNA‐Seq reads derived from N. benthamiana scion tissue did not map to the N. benthamiana reference genome v1.0, suggesting that rootstock‐derived RNAs may have mobilized into the scion as reported previously (Table S2; Li et al., 2022). To identify such mobile mRNAs, genomic DNA from Maxifort and Dro141TX tomato rootstocks were sequenced to establish gene sequence variations that occur between each rootstock and the N. benthamiana scion. A minimum of 250 million reads was obtained for each of two sequenced samples per rootstock. Reads were subsequently trimmed and mapped to the Solanum lycopersicum reference genome (SL3.0; accession GCF_000188115.4), resulting in 96% of each tomato rootstock genome having a 10× base coverage and 87% with 20× coverage. The fixed ploidy variant detection tool in CLC Genomics Workbench v. 20.0.4 was used to identify genome variants against the S. lycopersicum reference genome, including single and multinucleotide changes, deletions and insertions, and repeats with a probability >90%. A total of 271,769 nonsynonymous variants were identified in Dro141TX and 270,353 for Maxifort when compared to the tomato reference genome. By comparing the nonsynonymous variants from these two datasets, 25,409 Dro141TX‐specific variants and 23,982 Maxifort‐specific variants were identified with 246,736 variants being present in both datasets (Figure S3 and Table S3). The two rootstocks are thus more similar to each other than to the tomato reference genome.

The genomic sequences of Dro141TX and Maxifort were used to identify mobile rootstock transcripts present in the N. benthamiana scion (Bioproject PRJNA870056). Specifically, reads that did not map to the N. benthamiana genome were mapped to the S. lycopersicum reference genome (SL 3.0). Tomato‐mapped transcripts that had at least one read in all biological replicates were filtered and confirmed against the generated Dro141TX and Maxifort genome sequences. Reads per kilobase of transcript per million reads mapped (RPKM) values for each of the identified transcripts were averaged for the three biological replicates of each rootstock treatment and only transcripts with an RPKM above 100 in at least one treatment were selected for further analysis (Table S4). Results identified 489 tomato transcripts within N. benthamiana scion tissue. All 489 tomato transcripts were found in both Maxifort‐ and Dro141TX‐grafted N. benthamiana (Table S3). To further confirm the graft inducibility of these transcripts, tomato‐specific primers were designed to amplify a subset of the identified mobile transcripts within grafted N. benthamiana scion tissue (Table S4). Of the eight tested mobile tomato transcripts, reverse transcription (RT)‐PCR analysis confirmed the presence of five within grafted N. benthamiana scions (Figure S4). In addition, similar heterograft systems have been previously developed to investigate the movement of mRNAs over long distances (Notaguchi et al., 2015; Xia et al., 2018). Xia et al. (2018) used a tomato–N. benthamiana and potato heterograft system to identify 183 mobile mRNAs (Xia et al., 2018) while Notaguchi et al. (2015) identified 138 mobile mRNAs using an Arabidopsis and N. benthamiana system (Notaguchi et al., 2015; Xia et al., 2018). Consistent with this study, both Notaguchi et al. (2015) and Xia et al. (2018) also identified several ribosomal and translation‐associated mRNAs as graft mobile (Figure 4). Thus, similarities in transcript mobility exist between different heterograft systems.

FIGURE 4.

FIGURE 4

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Identification of mobile tomato transcripts within Nicotiana benthamiana grafted scions. Tomato transcripts from the N. benthamiana RNA‐Seq analysis with an RPKM (reads per kilobase of transcript per million reads mapped) above 100 in at least one treatment and with a two‐fold or greater difference between Maxifort (left panels) and Dro141TX (right panels) are shown (Table S2). Genome sequences derived from Dro141TX and Maxifort tomato rootstocks (Table S3 and Figure S2) were used to confirm the tomato transcripts within the N. benthamiana scion.

Although no rootstock‐specific transcripts with an RPKM value >100 were identified, there were specific transcripts in this category that displayed significant levels of enrichment in either Dro141TX‐ or Maxifort‐grafted tissue. Fold expression comparisons of shared transcripts derived from the two rootstocks identified nine tomato transcripts that displayed at least a two‐fold higher accumulation in Dro141TX‐grafted scions. Seven of the nine identified DroTX141‐enriched transcripts are derived from genes or gene families that have been linked to stress or disease‐resistance responses (Figure 4). For example, two major latex proteins (MLPs) were significantly enriched in Dro141TX‐grafted scions. MLPs have been associated with phloem expression and are known to bind hydrophobic compounds, including those involved in systemic acquired resistance (Fujita & Inui, 2021). The other two transcripts are linked to genes involved in abscisic acid signalling. Within Maxifort‐grafted scion tissues, 78 transcripts were found to have a two‐fold higher accumulation level. Of these 78 transcripts, 43 were associated with ribosome or translation, 18 were linked to metabolic or signalling processes, nine to nucleic acid interactions, four to chaperone functions, with the remaining having unknown functions. Thus, results from both mobile RNA analysis and RNA‐Seq are consistent with the observed phenotype, that is, higher levels of development‐related transcripts in scions grafted to Maxifort rootstocks and increased defence‐related transcripts present in scions grafted to Dro141TX.

The practice of grafting for enhanced crop production is well established. However, the molecular mechanisms responsible for conferring desired traits are poorly understood, making it difficult to predict phenotypic outcomes of specific rootstock–scion combinations. In this study, a heterograft system was developed and investigated for the ability to confer resistance against viral infection. The described tomato–N. benthamiana system represents a unique means to investigate the molecular components capable of conferring graft‐induced resistance. In particular, Dro141TX and Maxifort rootstocks are highly resistant to TMV infection yet only the Dro141TX confers a level of resistance to the normally susceptible N. benthamiana scion. This variation allows for the comparison of the genetic elements that are unique to each rootstock to identify elements capable of conferring resistance. The phenotypes and transcriptional alterations conferred by both Maxifort and Dro141TX were induced prior to infection, as observed in the scion RNA‐Seq analysis of uninfected tissue. Maxifort, which conferred larger leaves and greater growth to the N. benthamiana scion, induced the expression of a range of growth‐related genes. The resistance induced by Dro141TX was associated with the induction of stress‐ and defence‐associated genes within the N. benthamiana scion. Similar increases in stress‐related genes have also been observed in apple scions grafted to rootstocks that confer reduce susceptibility to the fire blight pathogen Erwinia amylovora (Jensen et al., 2003). This type of induced resistance may represent a form of graft incompatibility where specific physiological or genetic responses between the scion and rootstock induce stress and resistance responses. What these responses are remains to be determined. The transport of mRNAs from rootstock to scion was also modulated by the rootstock source, with Maxifort‐grafted scions enriched in translation‐ and development‐associated mobile transcripts and Dro141TX‐grafted scions enriched in defence‐ and stress‐associated mobile transcripts. The contrasting scion responses and corresponding phenotypes indicate that unique genetic differences between Maxifort and Dro141TX function to modulate the observed graft‐inducible resistance. Furthermore, the establishment of this heterograft system will permit additional investigations aimed at understanding the molecular rootstock–scion interactions and exchanges that are responsible for conferring resistance across the graft boundary.

CONFLICT OF INTEREST STATEMENT

All authors declare that they have no conflicts of interest.

Supporting information

FIGURE S1 Morphological differences within Nicotiana benthamiana scions. (a) Image of Maxifort, Estamino, and Dro141TX heterografts at 4 weeks after grafting. (b) Representative graft union. (c) Nicotiana benthamiana leaf sizes grafted on three different tomato rootstocks. Leaf and mid‐vein measurements were taken from the two to four leaves immediately above the graft union 3 weeks after grafting. Bars were averaged from 32 leaves. Bars connected by the same letter are not significantly different (t test, α = 0.05).

Click here for additional data file. (8MB, tif)

FIGURE S2 Correlation analysis of transcripts of three biological replicates from Nicotiana benthamiana scions grafted to Maxifort (M1, M2 and M3) and Dro141TX (D1, D2 and D3) tomato rootstocks. Two‐way comparison of scion transcripts grafted to Maxifort and Dro141TX used the 6745 identified differentially expressed genes, which were then subjected to Pearson correlation analysis.

Click here for additional data file. (7.7MB, tif)

FIGURE S3 Single‐nucleotide polymorphism density plots for the genomes of Dro141TX and Maxifort tomato rootstock. Single‐nucleotide variants (SNVs) obtained by comparison of Dro141TX or Maxifort genome sequences with the reference Solanum lycopersicum (SL3.0) genome. Colour scale represents number of SNVs per Mb of genome.

Click here for additional data file. (3MB, tif)

FIGURE S4 Reverse transcription‐PCR confirmation for the presence of mobile tomato transcripts within grafted Nicotiana benthamiana scion tissues. Tomato‐specific primers were developed based on the genomic sequences of Maxifort and Dro141TX (Table S4, Bioproject: PRJNA870056). These primers amplify transcript specific bands from Dro141TX and Maxifort tomato tissues as well as from N. benthamiana scion tissue grafted onto Dro141TX and Maxifort rootstocks but fail to amplify the transcript from nongrafted N. benthamiana tissue.

Click here for additional data file. (10.9MB, tif)

FIGURE S5 Western immunoblots for the detection of TMV coat protein (CP) within inoculated Nicotiana benthamiana scion leaves. Leaves of 3–4‐week‐old grafted scions were dusted with carborundum and inoculated with a solution of 0.2 μg/mL of purified TMV. Leaf punches (20 mg fresh weight) were taken at 2, 4 and 6 days postinoculation (dpi). Each lane represents two leaf punches taken from two independently inoculated scion leaves. Western immunoblots using TMV coat protein‐specific antibodies were run as previously described and coat protein band intensities determined using ImageJ (Dardick et al., 2000; Padmanabhan et al., 2008; Schneider et al., 2012)

Click here for additional data file. (15.5MB, tif)

TABLE S1 Primer sets used for TMV and mobile mRNA detection.

Click here for additional data file. (9.4KB, xlsx)

TABLE S2 RNA‐Seq parameters and analysis of Nicotiana benthamiana transcriptional changes.

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TABLE S3 Whole‐genome sequencing parameters and analysis of Maxifort and Dro141TX tomato rootstock.

Click here for additional data file. (6.3MB, xlsx)

TABLE S4 Identification of tomato mobile mRNAs within Nicotiana benthamiana scions.

Click here for additional data file. (4.9MB, xlsx)

ACKNOWLEDGEMENTS

This research was partially funded by the National Science Foundation Division of Integrative Organismal Systems grant number ISO‐1644713. Y.B.T. was additionally supported by the National Institutes of Health, training grant 5T32AI051967.

Kappagantu, M. , Brandon, M. , Tamukong, Y.B. & Culver, J.N. (2023) Rootstock‐induced scion resistance against tobacco mosaic virus is associated with the induction of defence‐related transcripts and graft‐transmissible mRNAs. Molecular Plant Pathology, 24, 1184–1191. Available from: 10.1111/mpp.13353

DATA AVAILABILITY STATEMENT

Supporting RNA‐Seq data were deposited in the Gene Expression Omnibus data repository (http://www.ncbi.nlm.nih.gov/geo/) under the reference number GSE211493 (accessions GSM6474711–GSM6474716). Tomato rootstock genome data were deposited in the Sequence Read Archive (https://submit.ncbi.nlm.nih.gov/subs/sra/) BioProject ID PRJNA870056.

REFERENCES

  1. Albert, R. , Kunstler, A. , Lantos, F. , Adam, A.L. & Kiraly, L. (2017) Graft‐transmissible resistance of cherry pepper (Capsicum annuum var. cerasiforme) to powdery mildew (Leveillula taurica) is associated with elevated superoxide accumulation, NADPH oxidase activity and pathogenesis‐related gene expression. Acta Physiologiae Plantarum, 39, 53. [Google Scholar]
  2. Antignani, V. , Klocko, A.L. , Bak, G. , Chandrasekaran, S.D. , Dunivin, T. & Nielsen, E. (2015) Recruitment of PLANT U‐BOX13 and the PI4K β1/β2 phosphatidylinositol‐4 kinases by the small GTPase RabA4B plays important roles during salicylic acid‐mediated plant defense signaling in Arabidopsis . The Plant Cell, 27, 243–261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bhandari, D.D. , Lapin, D. , Kracher, B. , von Born, P. , Bautor, J. , Niefind, K. et al. (2019) An EDS1 heterodimer signalling surface enforces timely reprogramming of immunity genes in Arabidopsis . Nature Communications, 10, 772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Castel, B. , Ngou, P.M. , Cevik, V. , Redkar, A. , Kim, D.S. , Yang, Y. et al. (2019) Diverse NLR immune receptors activate defence via the RPW8‐NLR NRG1. New Phytologist, 222, 966–980. [DOI] [PubMed] [Google Scholar]
  5. Cui, H.T. , Qiu, J.D. , Zhou, Y. , Bhandari, D.D. , Zhao, C.H. , Bautor, J. et al. (2018) Antagonism of transcription factor MYC2 by EDS1/PAD4 complexes bolsters salicylic acid defense in Arabidopsis effector‐triggered immunity. Molecular Plant, 11, 1053–1066. [DOI] [PubMed] [Google Scholar]
  6. Dardick, C.D. , Golem, S. & Culver, J.N. (2000) Susceptibility and symptom development in Arabidopsis thaliana to tobacco mosaic virus is influenced by virus cell‐to‐cell movement. Molecular Plant‐Microbe Interactions, 13, 1139–1144. [DOI] [PubMed] [Google Scholar]
  7. Davis, A.R. , Perkins‐Veazie, P. , Hassell, R. , Levi, A. , King, S.R. & Zhang, X.P. (2008) Grafting effects on vegetable quality. HortScience, 43, 1670–1672. [Google Scholar]
  8. Dongus, J.A. & Parker, J.E. (2021) EDS1 signalling: at the nexus of intracellular and surface receptor immunity. Current Opinion in Plant Biology, 62, 102039. [DOI] [PubMed] [Google Scholar]
  9. Fisher, K. & Turner, S. (2007) PXY, a receptor‐like kinase essential for maintaining polarity during plant vascular‐tissue development. Current Biology, 17, 1061–1066. [DOI] [PubMed] [Google Scholar]
  10. Fujita, K. & Inui, H. (2021) Review: biological functions of major latex‐like proteins in plants. Plant Science, 306, 110856. [DOI] [PubMed] [Google Scholar]
  11. Gonzalez, R. , Butkovi, A. , Rivarez, M.P.S. & Elena, S.F. (2020) Natural variation in Arabidopsis thaliana rosette area unveils new genes involved in plant development. Scientific Reports, 10, 17600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Guan, W.J. , Zhao, X. , Hassell, R. & Thies, J. (2012) Defense mechanisms involved in disease resistance of grafted vegetables. HortScience, 47, 164–170. [Google Scholar]
  13. Haroldsen, V.M. , Szczerba, M.W. , Aktas, H. , Lopez‐Baltazar, J. , Odias, M.J. , Chi‐Ham, C.L. et al. (2012) Mobility of transgenic nucleic acids and proteins within grafted rootstocks for agricultural improvement. Frontiers in Plant Science, 3, 39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jensen, P.J. , Rytter, J. , Detwiler, E.A. , Travis, J.W. & McNellis, T.W. (2003) Rootstock effects on gene expression patterns in apple tree scions. Plant Molecular Biology, 53, 493–511. [DOI] [PubMed] [Google Scholar]
  15. Kim, S.H. , Kwon, S.I. , Saha, D. , Anyanwu, N.C. & Gassmann, W. (2009) Resistance to the Pseudomonas syringae effector HopA1 is governed by the TIR‐NBS‐LRR protein RPS6 and is enhanced by mutations in SRFR1. Plant Physiology, 150, 1723–1732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kousik, C.S. , Mandal, M. & Hassell, R. (2018) Powdery mildew resistant rootstocks that impart tolerance to grafted susceptible watermelon scion seedlings. Plant Disease, 102, 1290–1298. [DOI] [PubMed] [Google Scholar]
  17. Lee, J.M. & Oda, M. (2002) Grafting of herbaceous vegetable and ornamental crops. Horticultural Reviews, 28, 61–124. [Google Scholar]
  18. Li, W.J. , Chen, S.M. , Liu, Y. , Wang, L.K. , Jiang, J.F. , Zhao, S. et al. (2022) Long‐distance transport RNAs between rootstocks and scions and graft hybridization. Planta, 255, 96. [DOI] [PubMed] [Google Scholar]
  19. Lu, X.H. , Liu, W.Q. , Wang, T. , Zhang, J.L. , Li, X.J. & Zhang, W.N. (2020) Systemic long‐distance signaling and communication between rootstock and scion in grafted vegetables. Frontiers in Plant Science, 11, 460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mohr, T.J. , Mammarella, N.D. , Hoff, T. , Woffenden, B.J. , Jelesko, J.G. & McDowell, J.M. (2010) The Arabidopsis downy mildew resistance gene RPP8 is induced by pathogens and salicylic acid and is regulated by W box cis elements. Molecular Plant‐Microbe Interactions, 23, 1303–1315. [DOI] [PubMed] [Google Scholar]
  21. Mudge, K. , Janick, J. , Scofield, S. & Goldschmidt, E.E. (2009) A history of grafting. Horticultural Reviews, 35, 437–493. [Google Scholar]
  22. Notaguchi, M. , Higashiyama, T. & Suzuki, T. (2015) Identification of mRNAs that move over long distances using an RNA‐Seq analysis of Arabidopsis/Nicotiana benthamiana heterografts. Plant and Cell Physiology, 56, 311–321. [DOI] [PubMed] [Google Scholar]
  23. Padmanabhan, M.S. , Kramer, S.R. , Wang, X. & Culver, J.N. (2008) Tobacco mosaic virus replicase‐auxin/indole acetic acid protein interactions: reprogramming the auxin response pathway to enhance virus infection. Journal of Virology, 82, 2477–2485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rubio, M. , Martinez‐Gomez, P. , Garcia, J.A. & Dicenta, F. (2013) Interspecific transfer of resistance to Plum pox virus from almond to peach by grafting. Annals of Applied Biology, 163, 466–474. [Google Scholar]
  25. Schneider, C.A. , Rasband, W.S. & Eliceiri, K.W. (2012) NIH Image to ImageJ: 25 years of image analysis. Nature Methods, 9, 671–675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sherman, B.T. , Hao, M. , Qiu, J. , Jiao, X.L. , Baseler, M.W. , Lane, H.C. et al. (2022) DAVID: a web server for functional enrichment analysis and functional annotation of gene lists (2021 update). Nucleic Acids Research, 50, W216–W221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shinohara, H. , Mori, A. , Yasue, N. , Sumida, K. & Matsubayashi, Y. (2016) Identification of three LRR‐RKs involved in perception of root meristem growth factor in Arabidopsis . Proceedings of the National Academy of Sciences of the United States of America, 113, 3897–3902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shivprasad, S. , Pogue, G.P. , Lewandowski, D.J. , Hidalgo, J. , Donson, J. , Grill, L.K. et al. (1999) Heterologous sequences greatly affect foreign gene expression in tobacco mosaic virus‐based vectors. Virology, 255, 312–323. [DOI] [PubMed] [Google Scholar]
  29. Spano, R. , Ferrara, M. , Gallitelli, D. & Mascia, T. (2020) The role of grafting in the resistance of tomato to viruses. Plants, 9, 1042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Spano, R. , Gallitelli, D. & Mascia, T. (2017) Grafting to manage infections of top stunting and necrogenic strains of cucumber mosaic virus in tomato. Annals of Applied Biology, 171, 393–404. [Google Scholar]
  31. Spano, R. , Mascia, T. , Kormelink, R. & Gallitelli, D. (2015) Grafting on a non‐transgenic tolerant tomato variety confers resistance to the infection of a Sw5‐breaking strain of Tomato spotted wilt virus via RNA silencing. PLoS One, 10, e0141319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Takahashi, H. , Shoji, H. , Ando, S. , Kanayama, Y. , Kusano, T. , Takeshita, M. et al. (2012) RCY1‐mediated resistance to Cucumber mosaic virus is regulated by LRR domain‐mediated interaction with CMV(Y) following degradation of RCY1. Molecular Plant‐Microbe Interactions, 25, 1171–1185. [DOI] [PubMed] [Google Scholar]
  33. Tsaballa, A. , Xanthopoulou, A. , Madesis, P. , Tsaftaris, A. & Nianiou‐Obeidat, I. (2020) Vegetable grafting from a molecular point of view: the involvement of epigenetics in rootstock‐scion interactions. Frontiers in Plant Science, 11, 621999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tuteja, N. , Tran, N.Q. , Dang, H.Q. & Tuteja, R. (2011) Plant MCM proteins: role in DNA replication and beyond. Plant Molecular Biology, 77, 537–545. [DOI] [PubMed] [Google Scholar]
  35. Wang, J.H. , Kucukoglu, M. , Zhang, L.B. , Chen, P. , Decker, D. , Nilsson, O. et al. (2013) The Arabidopsis LRR‐RLK, PXC1, is a regulator of secondary wall formation correlated with the TDIF‐PXY/TDR‐WOX4 signaling pathway. BMC Plant Biology, 13, 94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Xia, C. , Zheng, Y. , Huang, J. , Zhou, X.J. , Li, R. , Zha, M.R. et al. (2018) Elucidation of the mechanisms of long‐distance mRNA movement in a Nicotiana benthamiana/tomato heterograft system. Plant Physiology, 177, 745–758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zhang, H.Y. , Liu, X. , Zhang, X.Y. , Qin, N.N. , Xu, K.F. , Yin, W.H. et al. (2020) Phosphoinositide 3‐kinase promotes oxidative burst, stomatal closure and plant immunity in bacterial invasion. Frontiers in Plant Science, 10, 1740. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zhang, Q. & Xiao, S.Y. (2015) Lipids in salicylic acid‐mediated defense in plants: focusing on the roles of phosphatidic acid and phosphatidylinositol 4‐phosphate. Frontiers in Plant Science, 6, 387. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

FIGURE S1 Morphological differences within Nicotiana benthamiana scions. (a) Image of Maxifort, Estamino, and Dro141TX heterografts at 4 weeks after grafting. (b) Representative graft union. (c) Nicotiana benthamiana leaf sizes grafted on three different tomato rootstocks. Leaf and mid‐vein measurements were taken from the two to four leaves immediately above the graft union 3 weeks after grafting. Bars were averaged from 32 leaves. Bars connected by the same letter are not significantly different (t test, α = 0.05).

Click here for additional data file. (8MB, tif)

FIGURE S2 Correlation analysis of transcripts of three biological replicates from Nicotiana benthamiana scions grafted to Maxifort (M1, M2 and M3) and Dro141TX (D1, D2 and D3) tomato rootstocks. Two‐way comparison of scion transcripts grafted to Maxifort and Dro141TX used the 6745 identified differentially expressed genes, which were then subjected to Pearson correlation analysis.

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FIGURE S3 Single‐nucleotide polymorphism density plots for the genomes of Dro141TX and Maxifort tomato rootstock. Single‐nucleotide variants (SNVs) obtained by comparison of Dro141TX or Maxifort genome sequences with the reference Solanum lycopersicum (SL3.0) genome. Colour scale represents number of SNVs per Mb of genome.

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FIGURE S4 Reverse transcription‐PCR confirmation for the presence of mobile tomato transcripts within grafted Nicotiana benthamiana scion tissues. Tomato‐specific primers were developed based on the genomic sequences of Maxifort and Dro141TX (Table S4, Bioproject: PRJNA870056). These primers amplify transcript specific bands from Dro141TX and Maxifort tomato tissues as well as from N. benthamiana scion tissue grafted onto Dro141TX and Maxifort rootstocks but fail to amplify the transcript from nongrafted N. benthamiana tissue.

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FIGURE S5 Western immunoblots for the detection of TMV coat protein (CP) within inoculated Nicotiana benthamiana scion leaves. Leaves of 3–4‐week‐old grafted scions were dusted with carborundum and inoculated with a solution of 0.2 μg/mL of purified TMV. Leaf punches (20 mg fresh weight) were taken at 2, 4 and 6 days postinoculation (dpi). Each lane represents two leaf punches taken from two independently inoculated scion leaves. Western immunoblots using TMV coat protein‐specific antibodies were run as previously described and coat protein band intensities determined using ImageJ (Dardick et al., 2000; Padmanabhan et al., 2008; Schneider et al., 2012)

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TABLE S1 Primer sets used for TMV and mobile mRNA detection.

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TABLE S2 RNA‐Seq parameters and analysis of Nicotiana benthamiana transcriptional changes.

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TABLE S3 Whole‐genome sequencing parameters and analysis of Maxifort and Dro141TX tomato rootstock.

Click here for additional data file. (6.3MB, xlsx)

TABLE S4 Identification of tomato mobile mRNAs within Nicotiana benthamiana scions.

Click here for additional data file. (4.9MB, xlsx)

Data Availability Statement

Supporting RNA‐Seq data were deposited in the Gene Expression Omnibus data repository (http://www.ncbi.nlm.nih.gov/geo/) under the reference number GSE211493 (accessions GSM6474711–GSM6474716). Tomato rootstock genome data were deposited in the Sequence Read Archive (https://submit.ncbi.nlm.nih.gov/subs/sra/) BioProject ID PRJNA870056.

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