A single amino acid substitution in the movement protein enables the mechanical transmission of a geminivirus

Abstract Begomoviruses of the Geminiviridae are usually transmitted by whiteflies and rarely by mechanical inoculation. We used tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, to address this issue. Most ToLCNDV isolates are not mechanically transmissible to their natural hosts. The ToLCNDV‐OM isolate, originally identified from a diseased oriental melon plant, is mechanically transmissible, while the ToLCNDV‐CB isolate, from a diseased cucumber plant, is not. Genetic swapping and pathological tests were performed to identify the molecular determinants involved in mechanical transmission. Various viral infectious clones were constructed and successfully introduced into Nicotiana benthamiana, oriental melon, and cucumber plants by Agrobacterium‐mediated inoculation. Mechanical transmissibility was assessed via direct rub inoculation with sap prepared from infected N. benthamiana. The presence or absence of viral DNA in plants was validated by PCR, Southern blotting, and in situ hybridization. The results reveal that mechanical transmissibility is associated with the movement protein (MP) of viral DNA‐B in ToLCNDV‐OM. However, the nuclear shuttle protein of DNA‐B plays no role in mechanical transmission. Analyses of infectious clones carrying a single amino acid substitution reveal that the glutamate at amino acid position 19 of MP in ToLCNDV‐OM is critical for mechanical transmissibility. The substitution of glutamate with glycine at this position in the MP of ToLCNDV‐OM abolishes mechanical transmissibility. In contrast, the substitution of glycine with glutamate at the 19th amino acid position in the MP of ToLCNDV‐CB enables mechanical transmission. This is the first time that a specific geminiviral movement protein has been identified as a determinant of mechanical transmissibility.


| INTRODUC TI ON
Begomoviruses cause epidemics in many economically important crops worldwide, including cassava, cucurbits, eggplant, legumes, okra, pepper, potato, and tomato (Green et al., 2003;Varma and Malathi, 2003;Shih et al., 2007). The infection of cassava by African cassava mosaic virus (ACMV) causes annual economic losses of $1.9-2.7 billion in Africa (Legg and Fauquet, 2004). The infection of sugar beet by bean golden mosaic virus (BGMV) and tomato by tomato yellow leaf curl virus (TYLCV) also leads to a severe reduction in crop yields (Varma and Malathi, 2003).
One of the most important factors contributing to viral epidemics is the mode of transmission. Comprehensive knowledge of viral transmission mechanisms and their potential modifications during viral evolution may lead to the development of more effective preventive measures. Begomoviruses are usually transmitted by whiteflies (Bemisia tabaci). Similar to many phloem-limited viruses, including other geminiviruses and closteroviruses, begomoviruses are rarely mechanically transmissible by rub or sap inoculation (Garnsey et al., 1977;Wege and Pohl, 2007). Thus far, only approximately 20 out of 409 known begomovirus species have been reported to be mechanically transmissible to their natural hosts (Bock and Guthrie, 1978;Morales et al., 1990;Gilbertson et al., 1991;Garrido-Ramirez et al., 2000;Chatchawankanphanich and Maxwell, 2002;Usharani et al., 2004;Ajlan et al., 2007;Tsai et al., 2011).  (Fauquet et al., 2008). The DNA-A genome encodes proteins required for virus replication and encapsidation, whereas DNA-B encodes proteins responsible for viral movement between subcellular and intercellular compartments as well as pathogenicity (Krenz et al., 2012). Although the DNA-B genomes of some begomoviruses have been implicated in mechanical transmission (Levy and Czosenk, 2003;Wege and Pohl, 2007), the specific viral proteins involved in this aspect remain largely unknown.
Tomato leaf curl New Delhi virus (ToLCNDV) is a begomovirus with a bipartite genome. This virus could cause severe damage to many economically important crops in the families of Solanaceae and Cucurbitaceae, including tomato, pepper, potato, cucumber, melon, sponge gourd, and pumpkin (Padidam et al., 1995;Samretwanich et al., 2000;Usharani et al., 2004;Hussain et al., 2005;Ito et al., 2008;Khan et al., 2012;Lopez et al., 2015). ToLCNDV DNA-A has two open reading frames (ORFs; AV1 and AV2) on the viral-sense strand and four ORFs (AC1 to AC4) on the antisense strand. AV1 encodes a coat protein (CP) and AV2 encodes a pre-coat protein.
AC1, AC2, and AC3 encode a replication-associated protein (Rep), a transcriptional activator protein (TrAP), and a replication enhancer (REn), respectively. AC4 encodes a protein required for symptom development (Fondong, 2013). DNA-B has two ORFs: BV1 of the viral-sense strand encodes a nuclear shuttle protein (NSP) and BC1 of the antisense strand encodes a cell-to-cell movement protein (MP) (Jeske, 2009).
ToLCNDV is transmitted by whiteflies in nature. However, greenhouse tests have revealed that some ToLCNDV isolates can be mechanically transmitted by rub inoculation while others cannot. The ToLCNDV-OM isolate, originally identified from a diseased oriental melon plant, can infect oriental melon, pickling melon, bottle gourd, cucumber, zucchini, and luffa via mechanical sap inoculation (Chang et al., 2010). The ToLCNDV-potato isolate can also be transmitted to host plants via mechanical inoculation (Usharani et al., 2004). In contrast, the ToLCNDV-severe and ToLCNDV-cucumber isolates, which share a very similar genome organization and size with oriental melon and potato isolates, cannot be mechanically transmitted to their hosts (Padidam et al., 1995;Samretwanich et al., 2000). The divergent modes of mechanical transmissibility among ToLCNDV isolates may be due to the high genomic recombination and mutation rates of geminiviruses, which could lead to the emergence of a new isolate with different transmission abilities or host ranges (Chatchawankanphanich and Maxwell, 2002). New viral variants are often more pathogenic or can exhibit a wider host range than previously existing variants (Arguello-Astorga et al., 2007;Duffy and Holmes, 2008). For example, tomato yellow leaf curl Thailand virus (TYLCTHV) is mechanically transmissible and has spread widely to become a predominant strain in Taiwan (Tsai et al., 2011). In contrast, an endogenous tomato leaf curl Taiwan virus (ToLCTWV), which is not mechanically transmissible, has become less common in the region.
The mechanisms of the mechanical transmission of begomoviruses have not received much attention, because many of them are transmitted by whiteflies. The new ToLCNDV-CB isolate, recently identified from a cucumber plant with symptoms, shares high genome sequence similarity with the ToLCNDV-OM isolate. Unlike ToLCNDV-OM, the ToLCNDV-CB isolate cannot be transmitted to host plants by sap inoculation. In this study, genetic and pathological approaches were employed to identify the molecular determinants responsible for the mechanical transmission of ToLCNDV-OM. Gene swapping and point mutations revealed that the 19th amino acid residue at the N-terminus of the MP of DNA-B plays a critical role in mechanical transmission. The results also showed that the NSPcoding region plays no role in mechanical transmission.

| ToLCNDV isolates share high sequence similarity
The ToLCNDV-OM isolate, originally obtained from a diseased oriental melon, could be mechanically transmitted to its host plants. In contrast, the closely related ToLCNDV-CB isolate, originally collected from cucumber with symptoms, failed to infect Nicotiana benthamiana, oriental melon, or cucumber plants by mechanical sap inoculation.
Similar to ToLCNDV-OM, the ToLCNDV-CB isolate was found to have a bipartite genome consisting of DNA-A (2,738 nt) and DNA-B (2,695 nt). Pairwise sequence comparisons of the ToLCNDV-CB genome with the ToLCNDV-OM, ToLCNDV-severe (nonmechanically transmissible), and ToLCNDV-potato (mechanically transmissible) genome sequences available in GenBank were conducted to determine their genetic relationships. The whole-DNA-A-genome sequences of the ToLCNDV isolates shared from 92.8% to 96.5% nucleotide identity, and those of DNA-B shared from 84.4% to 92.3% nucleotide identity. The amino acid sequences encoded by DNA-A or DNA-B were highly similar, with the lowest similarity found among AC4 (88.1%-91.5%) of DNA-A and BC1 (88.7%-97.2%) of DNA-B (Table 1).
ToLCNDV-OM and ToLCNDV-CB showed 96.5% identity of their DNA-A sequences and 92.3% of their DNA-B genomes. In addition to their high sequence similarity, the two isolates were found to induce similar symptoms and present very similar host ranges ( Figure S1).

| The DNA-B genome of ToLCNDV-OM is required for mechanical transmissibility
Because ToLCNDV-CB was unable to infect oriental melon and N. benthamiana by mechanical sap inoculation, infectious clones of ToLCNDV-CB DNA-A and DNA-B amplified via the rolling circle amplification (RCA) method were independently constructed in the binary vector pCAMBIA1304 ( Figure 1) for further tests. Each clone contained two copies of the respective DNA sequences, and they were thus designated pCB2A (DNA-A) and pCB2B (DNA-B). The pCB2A and pCB2B infectious clones were successfully introduced into N. benthamiana, oriental melon, and cucumber by agroinoculation, resulting in visible symptoms (Table 2). Viral symptoms including curling, puckering, and mosaic appeared on N. benthamiana leaves 5-7 days post-inoculation (dpi) and on oriental melon and cucumber leaves 7-10 dpi. However, sap prepared from diseased N. benthamiana leaves inoculated with pCB2A and pCB2B failed to induce symptoms in any of the test plants after rub inoculation. The ToLCNDV-OM infectious clones pOM2A and pOM2B, constructed in our previous study (Chang et al., 2010), were also introduced into N. benthamiana, oriental melon, and cucumber by agroinoculation and induced symptoms at similar rates and with similar magnitudes to those induced by the ToLCNDV-CB clones. Sap prepared from the ToLCNDV-O-infected N. benthamiana leaves also resulted in symptoms on the test plants following rub inoculation at incidence rates ranging from 40% to 85% (Table 2). Viral DNA-A and DNA-B in the plant leaves with symptoms were monitored by PCR amplification using genomespecific primers, resulting in products with similar intensities.
To determine whether DNA-A or DNA-B plays a role in mechanical transmission, a combination of DNA-A and DNA-B from ToLCNDV-CB and ToLCNDV-OM was tested for infectivity via mechanical sap inoculation. When the test plants were infected with the pCB2A + pOM2B or pOM2A + pCB2B pseudorecombinants by agroinoculation, most developed symptoms at rates ranging from 83.3% to 100% (Table 2). When sap extracted from diseased N. benthamiana leaves infected with pCB2A + pOM2B was rub inoculated onto the test plants, many of them also developed symptoms at rates (37% to 73%) comparable to those in plants inoculated with pOM2A + pOM2B. In contrast, sap extracted from N. benthamiana

| The NSP gene of ToLCNDV-OM plays no role in mechanical transmissibility
Sequence alignment has shown that the NSP genes of ToLCNDV-OM and ToLCNDV-potato (both mechanically transmissible isolates) are 39 nt shorter than that of the ToLCNDV-severe isolate, which is not mechanically transmissible (Chang et al., 2010). To determine whether the addition of 39 nt at the 5′ end of the NSP gene of  The similarity of amino acids.
in a high level of infectivity in N. benthamiana and oriental melon (  To determine the roles of NSP and MP in mechanical transmission, two infectious clones, pCB1B and pOM1B, containing a single copy of the DNA-B genomes of the respective ToLCNDV isolates and 832-bp intergenic region (IR) tandem repeats, were generated ( Figure 3a). As controls, the three test species were agroinoculated with pCB2A + pCB1B or pOM2A + pOM1B, which resulted in a high disease incidence. Sap extracted from N. benthamiana infected with pOM2A + pOM1B, but not with pCB2A + pCB1B, also induced symptoms on the test plants (

| The ToLCNDV-OM MP is required for mechanical transmission
To determine the role of the ToLCNDV-OM movement protein in mechanical transmission, two infectious clones were generated. The  Figure S3). The results indicate that the 5′ but not the 3′ end of the MP of ToLCNDV-OM was required for mechanical transmissibility.

F I G U R E 1
The DNA-B genome of the tomato leaf curl New Delhi virus (ToLCNDV)-OM isolate is required for mechanical transmissibility.
(a) Physical maps of DNA infectious clones (pCB2A, pCB2B, pOM2A, and pOM2B) carrying the duplicated DNA-A or DNA-B genome of the ToLCNDV-CB or ToLCNDV-OM isolate. Genes encoded by DNA-A or DNA-B and the location of the intergenic region (IR) sequence are also indicated. The arrow indicates the direction of the transcription of each gene. (b) Images of Nicotiana benthamiana (10 days postinoculation, dpi) oriental melon, and cucumber plants (12 dpi) after mechanical inoculation with the wild-type virus (pCB2A + pCB2B and pOM2A + pOM2B) or pseudorecombinant viruses (pCB2A + pOM2B and pOM2A + pCB2B) developing viral symptoms (O) or exhibiting no symptoms (X). The viral inoculum used for mechanical sap inoculation was prepared from diseased N. benthamiana after agroinoculation with an appropriate combination of the clones. (c) PCR detection of virus accumulation in the leaves after mechanical inoculation with pCB2A + pCB2B (lanes 1, 5, and 9), pOM2A + pOM2B (lanes 2, 6, and 10), pCB2A + pOM2B (lanes 3, 7, and 11), and pOM2A + pCB2B (lanes 4, 8, and 12  c The sap was prepared from the agroinoculated N. benthamiana and used as inoculum for mechanical inoculation. d The symbol "-" indicates that infectivity was not determined.

| A single amino acid in the ToLCNDV-OM MP determines mechanical transmissibility
Sequence alignment of ToLCNDV-OM and ToLCNDV-CB MPs revealed four amino acid differences (3rd, 6th, 8th, and 19th) in the N-terminal region (Figure 4a). Site-directed mutagenesis was performed to identify the specific amino acid residue(s) that are essential for mechanical transmissibility. Eight infectious clones, each containing a specific mutation of these four amino acids, were constructed and tested for infectivity ( Figure 5). Agroinoculation and rub inoculation assays revealed that infectious clones pCB1B MP(3I→T) ,

| Nonmechanically transmissible viruses fail to accumulate at detectable levels in plants after sap inoculation
Oriental melon plants that were inoculated with ToLCNDV infectious clones developed visible symptoms at 7-10 dpi. After

| D ISCUSS I ON
Begomoviruses are often transmitted by whiteflies and are rarely mechanically transmissible by rub or sap inoculation (Wege and Pohl, 2007).
Only a few known species in the Begomovirus genus have been reported to be mechanically transmissible to their natural hosts (Gilbertson et al., 1991;Usharani et al., 2004;Tsai et al., 2011). ToLCNDV is a begomovirus with a bipartite (DNA-A and DNA-B) genome (Padidam et al., 1995).
In the present study, genetic and pathological approaches were applied to identify the molecular determinants involved in mechanical transmissibility in a ToLCNDV-OM isolate originally collected from a diseased oriental melon. Unlike ToLCNDV-OM, the closely related ToLCNDV-CB isolate is not mechanically transmissible to oriental melon, cucumber or Although NSP has been reported to be required for begomovirus F I G U R E 3 The tomato leaf curl New Delhi virus (ToLCNDV)-OM movement protein (MP), but not nuclear shuttle protein (NSP), is required for mechanical transmission. (a) Schematic illustration of the pCB1B and pOM1B infectious clones carrying a single copy of the DNA-B genome of the CB or OM isolate, respectively. Four additional constructs, pCB1B::OM NSP , pOM1B::CB NSP , pCB1B::OM MP , and pOM1B::CB MP , were produced by swapping the BV1 (encoding NSP) or BC1 (encoding MP) gene between two ToLCNDV isolates. The primers used for construction are indicated above each genome. (b) Nicotiana benthamiana, oriental melon, and cucumber plants after mechanical inoculation with pCB2A + pCB1B, pOM2A + pOM1B, or other infectious clones, as indicated, developed viral symptoms (O) or exhibited no symptoms (X) at 10-12 days post-inoculation. The viral inoculum used for mechanical sap inoculation was prepared from N. benthamiana with symptoms after agroinoculation with an appropriate combination of the clones. Symptoms were observed exclusively in plants inoculated with the constructs carrying the ToLCNDV-OM MP gene. (c) PCR detection of viral DNA in plants after mechanical inoculation with pCB2A + pCB1B (lanes 1, 7, and 13), pCB2A + pCB1B::OM NSP (lanes 2, 8, and 14), pCB2A + pCB1B::OM MP (lanes 3, 9, and 15), pOM2A + pOM1B (lanes 4, 10, and 16), pOM2A + pOM1B::CB NSP (lanes 5, 11, and 17), or pOM2A + pOM1B::CB MP (lanes 6, 12, and 18). Specific primers were used to amplify the NSP, MP, or intergenic region (IR) fragment from the OM or CB isolate. Amplicons were obtained only from plants inoculated with the constructs carrying the ToLCNDV-OM MP gene virulence (Hussain et al., 2005;Zhou et al., 2007), a role of this gene in mechanical transmissibility was ruled out by testing the infectious  (Oldfield et al., 2005;Cheng et al., 2007). Disordered protein regions, characterized by the lack of a fixed tertiary structure, have been proposed to be involved in many biochemical functions, including protein binding and recognition (Dunker et al., 2002;Xue et al., 2010). PONDR analyses suggested that the N terminus of ToLCNDV-OM MP is a disordered region; a switch from glutamate to glycine at the 19th amino acid position changes the prediction to an ordered region. Moreover, the N terminus of ToLCNDV-CB MP is predicted to be an ordered region; however, the sequence is predicted to be a disordered region after the substitution of glycine with glutamate at the 19th amino acid.
In addition to MP, CP may be involved in mechanical transmissibility in ToLCNDV-OM as demonstrated in BGMV, another member of the Begomovirus genus. The BGMV CP has been demonstrated to be required for both mechanical and whitefly transmissions (Azzam et al., 1994). A BGMV mutant with a defective CP fails to induce symptoms on sap inoculation, but an infection develops after the mutant is biolistically bombarded into leaf cells because biolistic inoculation can deliver the virus directly into the host nucleus.
Thus, CP is required for the localization of BGMV to the nucleus.
Intriguingly, the CP of two geminiviruses, mungbean yellow mosaic virus (MYMV) and ACMV, have been reported to contain a nuclear localization signal (NLS) (Unseld et al., 2001;Guerra-Peraza et al., 2005), suggesting the involvement of CP in nuclear localization.

Therefore, the difference in mechanical transmissibility between
ToLCNDV-OM and ToLCNDV-CB may result from the interactions of CP and MP, which was shown not to be required for the mechanical transmission of ToLCNDV in the present study.
MP has also been implicated in intra-and intercellular trafficking by interacting with host proteins (Jeske, 2009), which could lead to different modes of transmission as seen in different ToLCNDV isolates. AbMV MP can interact with host kinases and affect the development of symptoms and the accumulation of viral DNA in host cells (Kleinow et al., 2009). Studies have identified histone H3 (Zhou et al., 2011), synaptotagmin (SYTA) (Lewis and Lazarowitz, 2010), and the 70 kDa heat shock protein cpHSC70-1 (Krenz et al., 2010)  Viral MPs are required for both cell-to-cell and long-distance movements. Studies on AbMV, another geminivirus, have suggested that a so-called "pilot domain" (amino acid residues from 1 to 49) of F I G U R E 4 The 5′-end of the movement protein (MP) of the tomato leaf curl New Delhi virus (ToLCNDV)-OM is required for mechanical transmissibility. (a) Schematic illustration of the pCB1B and pOM1B infectious clones showing eight different amino acid residues in MP between the ToLCNDV-OM and CB isolates. Four different amino acids (3rd, 6th, 8th, and 19th) were found at the N terminus, and four other amino acids (193rd, 225th, 233rd, and 262nd) were found at the C terminus of MPs. Two clusters of different amino acids were separated via the PstI restriction enzyme recognition site. Four recombinant constructs, pCB1B::OM 5′MP , pCB1B::OM 3′MP , pOM1B::CB 5′MP , and pOM1B::CB 3′MP , were produced by exchanging the 5′MP-intergenic region (IR) fragment (455 bp) and the 3′-end MP fragment (817 bp) between two isolates using EcoRV, PstI, and/or HindIII cleavage. (b) Nicotiana benthamiana, oriental melon, and cucumber plants after mechanical inoculation with the infectious clones, either developed viral symptoms (O) or exhibited no symptoms (X) at 10-12 days postinoculation. The viral inoculum used for mechanical sap inoculation was prepared from N. benthamiana with symptoms after agroinoculation with an appropriate combination of the clones. Symptoms were observed only in plants inoculated with the constructs carrying the 5′ region of the ToLCNDV-OM MP gene. (c) PCR detection of the NSP-MP DNA fragment in plants after mechanical inoculation with pCB2A + pCB1B (lanes 1, 7, and 13), pCB2A + pCB1B::OM 5′MP (lanes 2, 8, and 14), pCB2A + pCB1B::OM 3′MP (lanes 3, 9, and 15), pOM2A + pOM1B (lanes 4, 10, and 16), pOM2A + pOM1B::CB 5′MP (lanes 5, 11, and 17), and pOM2A + pOM1B::CB 3′MP (lanes 6, 12, and 18). Amplicons were obtained only from plants inoculated with the constructs carrying the 5′ end of the MP of ToLCNDV-OM MP is responsible for the transportation of the virus to cell periphery or nucleus (Zhang et al., 2002). Two models have been proposed to explain the functions of MP in geminiviruses (Rojas et al., 2005;Jeske, 2009). For phloem-limited begomoviruses such as AbMV and cabbage leaf curl virus (CaLCuV), MP is thought to attach to the NSP-bound viral DNA and form a complex that localizes along the plasma membrane or is transferred to adjacent cells through plasmodesmata. For mesophyll-invading begomoviruses, MP is thought to take over viral DNA from NSP after being exported from the nucleus to the cytoplasm and deliver it to adjacent cells. Most nonmechanically transmissible begomoviruses have been reported to be phloem limited because they can be introduced into the phloem F I G U R E 5 A single glutamate residue in the tomato leaf curl New Delhi virus (ToLCNDV)-OM movement protein (MP) determines mechanical transmissibility. Nicotiana benthamiana, oriental melon, and cucumber plants after mechanical inoculation with pCB2A paired with pCB1B MP(3I→T) , pCB1B MP(6D→E) , or pCB1B MP(8V→M) carrying a single amino acid substitution at the 3rd, 6th or 8th position of the MP of the CB isolate did not develop visible symptoms (X). However, plants that were mechanically inoculated with pCB2A and pCB1B MP(19G→E) carrying a substitution mutation at the 19th amino acid, resulting in a change from glycine to glutamate, developed viral symptoms (O). Plants that were mechanically inoculated with pOM2A and pOM1B MP(3T→I) , pOM1B MP(6E→D) , or pOM1B MP(8M→V) also developed visible symptoms. In contrast, plants that were mechanically inoculated with pOM2A and pOM1B MP(19E→G) , carrying a single amino acid substitution at the 19th amino acid, resulting in a change from glutamate to glycine, failed to develop symptoms. The viral inoculum used for mechanical sap inoculation was prepared from N. benthamiana with symptoms after agroinoculation with an appropriate combination of the clones. Abbreviations for amino acids: G, glycine; E, glutamate; I, isoleucine; T, threonine; D, aspartic acid; V, valine; M, methionine For systemic infection, ToLCNDV must be loaded into the phloem sieve tube system for long-distance movement. ToLCNDV-OM is able to propagate and travel through mesophyll cells, the bundle sheath, and eventually the phloem after sap inoculation, which is very rare for a geminivirus (Wang et al., 1996). In contrast, ToLCNDV-CB apparently has to rely on the stylet of its insect vector to reach the phloem. After sap inoculation, ToLCNDV-CB is unable to move beyond its inoculation foci, for which there may be many

| Characteristics and origins of ToLCNDV isolates
ToLCNDV-OM was originally isolated from a diseased oriental melon  (Chang et al., 2010).
A 39-nt fragment was amplified by PCR using the primers

| Agroinoculation and mechanical inoculation
Infectious clones were individually transformed into Agrobacterium tumefaciens C58 by using a GenePulser II electroporator (Bio-Rad).
Agroinoculation was conducted according to the procedure de-  Technology) and buffer. The conditions used for amplification were as follows: one cycle at 95 °C for 5 min followed by 35 cycles of 95 °C for 1 min, 50-67 °C depending on the specific primer pairs used (Table S1) for 1 min and 72 °C for 1 min, with a final cycle at 72 °C for 10 min. The PCR products were subjected to electrophoresis in a 0.8% agarose gel. Sequencing was performed using an ABI PRISM 3730 automatic DNA sequencer (Applied Biosystems) available at the Biotechnology Center, National Chung Hsing University (Taichung, Taiwan).

| Southern blot hybridization
After mechanical inoculation, the cotyledons of oriental melon were harvested at 0, 5, 10, and 14 dpi and subjected to DNA isolation.
DNA (5 μg) was subjected to electrophoresis in a 1% agarose gel and transferred to a nylon membrane (PerkinElmer) via the alkaline transfer method, and hybridization was performed on the full-length MP of ToLCNDV-OM. The probe was labelled by α-[ 32 P]dATP. The hybridization and detection of the probe were performed according to the methods described by Kon et al. (2003). DNA isolated from the apical leaves of oriental melon agroinoculated with ToLCNDV-OM (pOM2A + pOM1B) or ToLCNDV-CB (pCB2A + pCB1B) was used as a positive control.

| In situ hybridization
Tissue fixation and in situ hybridization were performed following the protocol described by Takeda et al. (2011) with some modifications. N. benthamiana leaves at 5 dpi were collected, cut into small pieces, and fixed in solution containing 50% ethanol, 5% acetic acid, and 5% formaldehyde (FAA) for 2 hr at room temperature.

| Predictor of natural disordered regions
The PONDR within the movement protein sequences of ToLCNDV-OM and ToLCNDV-CB was performed by using the PONDR VL-XT predictor available at http://www.pondr.com/.

ACK N OWLED G EM ENTS
We are grateful to Dr Kuang-Ren Chung for his critical review of this manuscript. We also thank Dr Shi-Dong Yeh for valuable discussion. This work was supported by grants from the Ministry of Science and Technology (NSC 101-2313-B-005-039-MY3 and MOST 105-2313-B-005-019-MY3), Executive Yuan, Taiwan. The authors have no conflicts of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.