A review of factors affecting the success of geminivirus infectious clones

Vol.:(0123456789) Plant Cell Reports (2025) 44:189 https://doi.org/10.1007/s00299-025-03560-4 REVIEW A review offactors affecting thesuccess ofgeminivirus infectious clones S.R.Möller1 · M.N.Maruthi1 Received: 20 March 2025 / Accepted: 23 June 2025 / Published online: 4 August 2025 © The Author(s) 2025 Abstract Key message In this review, we provide a summary of factors that affect the successful infection of geminivirus clones in plants to enable the greater understanding of plant–virus interactions. Abstract Geminiviruses are single-stranded DNA viruses that can cause significant losses in economically important crops worldwide. Considerable efforts have been made to study the geminiviruses in detail, which has resulted in the construction of many infectious clones for the vast diversity of geminiviruses. In laboratory conditions, agrobacterium or occasionally biolistic methods are used to deliver viral DNA to the plant cell. However, not every delivered viral DNA will develop into an infection due to several reasons. In this manuscript, we review the factors that affect the success of geminivirus infectious clones. Factors affecting virus infection including the methods of inoculating invitro-generated viral DNA constructs are often neglected, leading to failed virus infections and drawing wrong conclusions. Deciding exactly where on the plant to inoculate, what age of plant, and what agrobacterium strain are all examples of variables which may influence an infection. We find that stem injections of agrobacterium into young seedlings with an optical density at 600nm (OD600) in the 0.1–0.3 range are an optimal starting point for studies. This review will provide a thorough compilation of inoculation methods and use this to discuss the deeper mechanisms at play during the initial infection of plants with geminivirus infectious clones. Graphical abstract Keywords Agrobacterium infiltration· Begomovirus· Plant immunity· Biolistic· Virus transmission Communicated by Wusheng Liu. Extended author information available on the last page of the article Plant Cell Reports (2025) 44:189189 Page 2 of 22 Introduction Geminiviruses are a group of plant viruses that can infect and cause diseases on a wide range of both dicot and monocot species in the tropics, causing high economic losses, sometimes up to 100%, at least since the 1970s, in several food, fibre, fruit and industrial crops such as beans, cassava, cotton, maize, tobacco and many vegetables (Rojas etal. 2018). Family Geminiviridae is the largest group of single-stranded DNA viruses infecting plants, currently containing about 520 distinct species. The main genus Begomovirus in the family Geminiviridae contains at least 445 distinct species, whilst the second largest genus Mastrevirus contains about 45 species. The remaining 30 species are spread out amongst the 15 other genera with only a few viruses in each (Fiallo-Olivé etal. 2021; Roumagnac etal. 2022). Geminiviruses derive their name from their coat protein (CP) structure which takes the form of twinned icosahedral particles. Each particle contains a single copy of a single-stranded circular DNA molecule of around 2600 to 3200 base pairs in length (Hesketh etal. 2018). Most geminiviruses are monopartite with a single-genome DNA-A containing 4 to 8 overlapping open reading frames (ORFs) or genes. However, some begomoviruses are bipartite and have an additional DNA-B component, which codes for two ORFs. In addition, smaller satellite DNAs are found in association with some geminiviruses (Zhou 2013; Briddon etal. 2018). Geminiviruses confine to the phloem and are often found in fully differentiated cells; hence, the virus must carry out some reprogramming of the cell cycle to acquire the factors needed for replication (Hanley-Bowdoin etal. 2013). The genome structure of geminiviruses (Fig.1) differs slightly between the various genera. Common to them is the presence of an intergenic region containing the iteron sequences, bi-directional promoters and a stemloop structure required for DNA replication. The principle of making infectious clones remains the same for all geminiviruses; the virus must contain slightly more than one unit length which encompasses two stem loops, which allows it to reform into circular DNAs (Saad etal. 2021). This also extends to beta-, alphaand delta-satellites as well as to other circular DNA viruses such as nanoviruses. Among the ORFs, geminiviruses encode a replication protein (Rep), which does not have its own DNA polymerase activity but instead recruits host polymerases to replicate the virus. The Rep cleaves the, mostly, conserved nonanucleotide motif TAA TAT TAC. The Rep cleaves a single strand of DNA, favouring the viral sense strand, and facilitates the rejoining to initiate virus replication (Laufs etal. 1995). Replication was initially considered to be rolling circle replication where the Rep binds to iteron sequences in the origin of replication (ori) and recruits host polymerases to continuously copy the genome (Saunders etal. 1991). Later, electron microscopy and further analysis of the replication intermediates suggested a recombinationdependent virus replication method, whereby heterologous double-stranded linear fragments are produced which then recombine to form circular DNA (Jeske etal. 2001). This discussion is reviewed in depth in Bonnamy etal. (2023). For geminiviruses, the Rep proteins and virus factors for cell cycle reprogramming are normally transcribed from the complementary sense strand, whilst the CP and proteins involved in cell-to-cell movement are normally transcribed from the viral sense strand. Most geminiviral proteins carry out multiple functions by interacting with several distinct host factors to promote a cellular environment supporting viral infection, suppress silencing mechanisms and facilitate movement. This is reviewed in Fondong (2013) and Fiallo-Olivé etal. (2021). As new techniques have made cloning viral vectors easy (Ferro etal. 2019; Yıldırım etal. 2023), achieving efficient initial infections in relevant crop plants has often remained a possibility (Liu etal. 2021; Zhang etal. 2024; Kavalappara etal. 2024; Kumari etal. 2024). Geminivirus particles are naturally introduced to plants by insects, which have acquired the virus from feeding on infected plants. The difficulties, however, is the maintenance and handling of insects in laboratory conditions, which give rise to issues with insect containment and contamination. Alternative methods of virus inoculations, therefore, using A. tumefaciens (henceforth referred to as agrobacterium) wounding, and/ or particle bombardment have been sought. Collectively, geminiviruses infect a vast range of plant hosts, but individual viruses can have a narrower host range. Even minor variations within the virus can make it ineffective in a host plant (Vo etal. 2023), and variants of plants form the basis of resistant cultivars. One might anticipate a geminivirus to produce infections in all the inoculated plants of a susceptible species. Yet in many cases, only some of them develop an infection following the inoculation and thus creating uncertainty on the use of infectious clones in experiments. Having a high percentage of inoculated plants become symptomatic is of interest to, for example, screen for resistant crop varieties and understand the host range of viruses. Initial infection represents a decisive moment in viral infection deciding the resistance vs. susceptibility, which make observations of the factors driving the initial infection even more relevant. Plant Cell Reports (2025) 44:189 Page 3 of 22 189 A large number of studies have used infectious clones; however, effective comparison of the disease incidences between the studies is difficult as ultimately variations in cultivar and virus strains are the main determinants, which are often not the focus of the investigations. In addition, many factors, such as the age of the plant, light and environmental conditions, are often unrecorded and unlikely to follow a prevalent standard. However, a few experiments have optimised various parameters, such as density of agrobacterium or age of plant, and reviewing this limited evidence gives us guidelines to optimise conditions for new experiments. Obtaining high rates of successful infections from agrobacterium or biolistic inoculations will give high levels of confidence in the data we generate, which is crucial for subsequent research. Whitefly transmission Different members of Geminiviridae are spread by different insect vectors (Wang and Blanc 2021). Within the begomovirus group, different species of whiteflies can have preference for specific begomoviruses due to their feeding preference for certain host plants and the subsequent co-adaptation (Maruthi etal. 2002; Campbell etal. 2023). The geminivirus-transmitting insects all feed on plants by piercing the leaf with their mouthpiece and sucking the sap from the vascular system and either acquiring or delivering the virus in so doing. Insect transmission depends on several factors including the amount of virus carried by the insect and the amount of feeding (Wang etal. 2022a; Janssen etal. 2022). A single whitefly fully loaded on begomovirus Fig. 1 Typical structure of geminiviral genomes (only begomovirus and mastrevirus genomes shown as examples). The common region (CR) contains replication origin. Rep replication protein—in mastrevirus, the splice variant RepA is present which takes on additional functions similar to TrAP and REn (Diamos and Mason 2019; Watanabe and Ugaki 2021); TrAP Transcription activator protein; REn replication enhancer; SIR Small intergenic region which acts as terminator; CP coat protein; Pre-CP pre-coat protein which also aids in movement; V1 coat protein; AC4 is a virulence factor; MP movement protein; NSP nuclear shuttle protein; SCR satellite conserved region; βC1 satellite pathogenicity factor; A-rich adenine rich region. Reading frames AC5, AC6 and AV3 are additional small reading frames potentially found in begomoviruses with additional putative functions e.g. in silencing suppression(Gong etal. 2021; Wang etal. 2022b) Plant Cell Reports (2025) 44:189189 Page 4 of 22 can deposit viral DNA, into the leaf, in the range of nanograms (Roy etal. 2021). Comparing this to agrobacterium or mechanical inoculation may not be meaningful as there is no accounting for the amount of viral DNA which makes it into cell nuclei by these methods. In insect transmission, the CP not only assists with entry of the single-stranded DNA (Krichevsky etal. 2006) but can also have functions that are critical for the establishment of infection (Vo etal. 2023). The insect vectors are not only obedient carriers of the virus as they feed on plants, but also deliver effectors to the plant that assist the insect in feeding whilst also modifying the plant immune signalling. In return, the virus can make plants easier to feed on (Naalden etal. 2021; Ray and Casteel 2022). Whiteflies therefore represent the most certain way of geminivirus transmission. The malvastrum leaf curl Guangdong virus (MLCuGdV), for example, was unable to initiate infection in its natural host Malvastrum coromandelianum via agroinfection, whilst whiteflies were able to transmit (Wu etal. 2007; Guo etal. 2008). However, whitefly colonies are difficult to maintain, and they are not allowed to be maintained in some countries due to quarantine reasons. Developing virus infectious clones therefore is the easier way of researching geminiviruses on aspects that do not require transmission by the insect vectors. The plant immunity N. benthamiana is commonly used as the first host for testing virus infectivity owing to its susceptibility to plant viruses, which is due to deficiencies in its silencing pathways (Yang etal. 2004; Goodin etal. 2008; Fujiuchi etal. 2016; Bally etal. 2018). It also helps that it is easy to fill the apoplastic space of N. benthamiana with agrobacterium suspension which allows it to reach the phloem area (Guan and Zhou 2006). In most other plants, leaf infiltration is far less efficient due to their rigid leaf morphologies. Although it is not a limitation on agrobacterium to deliver the DNA in the wounded area, it is the various mechanisms of gene silencing, which prevents initial expression and thereby virus infection (Wroblewski etal. 2005; Dunoyer etal. 2006; Bilichak etal. 2014; Zhang etal. 2020; Azizi-Dargahlou and Pouresmaeil 2023). Multiple plant immunity pathways silencing the T-DNA carrying virus constructs are reviewed (Vaucheret 2022; Roca Paixao and Déléris 2025). In brief, once ‘detected’ small double-stranded RNAs (dsRNAs) are produced from the invading DNA. These are converted by DICER ribonucleases into small interfering RNAs and loaded onto the RNA-induced silencing complexes which targets the corresponding DNA sequence for methylation and the formation of heterochromatin preventing virus protein expression, or targeting the expressed viral mRNAs for cleavage, removing them. That is, of course canonical and simplified, as viruses have ways of countering the host silencing which has evolved secondary pathways to circumvent it (Gupta etal. 2021; Zhang etal. 2023). What is noteworthy here regarding the initial geminivirus infection is the systemic spread of the RNA interference (RNAi) silencing response through systemic movement of siRNA in the plant (Molnar etal. 2010; Qin etal. 2012; Zhang etal. 2019; Sanan-Mishra etal. 2021). The activation of RNAi negatively affects geminivirus infectious clone (Kumar etal. 2017; Sangwan etal. 2023). This could contribute to an activation of siRNA mechanism in wider leaf area as it would occur during leaf infiltrations. Having a large number of cells with active siRNA could lead to the overall abortion of virus infection. Therefore, leaf infiltration of agrobacterium is seemingly less effective than, for example, stem injections where the virus is potentially introduced into phloem tissues, which are the preferred by geminiviruses. The mechanism ofDNA transfer inagroinfections For the infectious clones, the viral DNA is delivered as the dimer and is then eventually converted to a circular DNA, which makes the details of agrobacterium DNA delivery worth considering. As nature’s natural genetic engineer, the bacterium inserts a transfer-DNA (T-DNA), held in its tumour inducing plasmid (Ti-plasmid), encoding genes which when inserted into the plant genome induces tumour formation and engineer the plant to produce the opines only agrobacterium can metabolise. The Ti-plasmid are about a quarter million base pairs large and, in addition to the T-DNA, also contain many of the virulence genes involved in T-DNA delivery. Although, most agrobacterium strains used for plant transformation have had their tumour inducing native T-DNA removed. As the agrobacterium T-DNA is selected for transfer by a short left and right border sequence, the binary plasmids can transfer the geminivirus as T-DNA, by inserting it between the border sequences. One sidenote of geminivirus DNA in agrobacterium is that agrobacterium will also express the Rep protein which then creates circular DNAs inside the agrobacterium by its nicking and re-ligation activity (Selth etal. 2002) (Fig.2c). Agrobacterium delivers multiple copies of T-DNA through multiple copies of the binary plasmid (Oltmanns etal. 2010) and thus deliver multiple copies of viruses into a plant cell to initiate virus infection. The agrobacterium enzyme VirD2 nicks the Ti-plasmid DNA at the right T-DNA border and covalently binding itself to the 5’-end pulling out a singlestranded DNA (Jasper etal. 1994). Agrobacterium coats the T-DNA with the VirE2 protein which protects it on its way to the plant nucleus (Fig.2b) (Christie 2004; Li etal. 2020). Plant Cell Reports (2025) 44:189 Page 5 of 22 189 The agrobacterium essentially creates a channel, the T-pilus into the plant cell to transfer both the DNA and agrobacterium virulence proteins (Nester 2015). Whilst discussion is ongoing as to the exact mechanisms of T-DNA integration into the plant genome (Gelvin 2021; Thomson etal. 2024), integration is not needed as part of the transient expression, like for geminiviruses (Singer 2018). The single-stranded DNA is converted to double-stranded DNA with some ending up as various circular “T-Circle” T-DNAs. Often two T-DNAs will fuse tail–tail first, as VirD2 remains covalently bound, before becoming circular through a recombination process (Fig.2e) (Singer etal. 2012; Gelvin etal. 2022). Getting the geminiviral T-DNA dimer into the nucleus is only one step, to become a virus infection, the geminivirus must be ejected from the T-DNA (Fig.2f). The ejection of the circular viral DNA from the dimer is, like the replication mechanism, not fully understood, as these could also escape through recombination-dependant means (Bonnamy etal. 2023). In mechanical inoculation, viral DNA also retains infectivity as linear DNA, because the host repair Fig. 2 Chart describing the journey of geminiviral DNA from agrobacterium binary vector to circular DNA in the plant cell nucleus. a. VirD2 in conjunction with VirD1 nicks DNA, VirD2 binds to the right border (RB) of the T-DNA, excising a single-stranded DNA from the binary T-DNA vector. The DNA contains an infectious dimer of a geminivirus. b. The nascent T-DNA is coated with VirE2 and then transferred through the T-pilus T4SS channel into the plant cell cytosol along with other virulence factors. c. Regardless of the two previous steps residual expression of Rep by the agrobacterium allows circular DNAs of the virus to form within the agrobacterium. This circular DNA plays no part in infection (Grimsley etal. 1987). d. VirD2 and VirE2 interact with the nuclear pore complex to import the T-DNA to the plant cell nucleus. e. The T-DNA is subjected to the plant DNA repair mechanisms turning the single-stranded DNA into various double-stranded forms and the Rep is expressed. f. The Rep will initiate replication and liberate single circular virus genome virus from the T-DNA, the order of which is unclear Plant Cell Reports (2025) 44:189189 Page 6 of 22 mechanisms readily re-ligates it to a circular molecule (Lapidot etal. 2007). Inoculation with plasmid DNA containing a single copy of the virus should not be infectious as the plasmid backbone interrupts the virus sequence. However, monomeric clones of pepper huasteco yellow vein virus was infectious as a plasmid if the plasmid backbone was inserted into the coat protein, with the resulting systemic virus infection having reverted to the natural size. The coat protein is not necessary for its replication and thus the entire plasmid must undergo replication, with recombination eventually ejecting the intervening plasmid sequence (Bonilla-Ramírez etal. 1997; Lapidot etal. 2007). This is particularly true in geminiviruses as they appear to be selective towards their native size (Bisaro 1994). The recent discovery of superinfection exclusion mechanism complicates the matter somewhat because it is possible to speculate on some mechanisms where only a few initial copies enter rolling circle replication (Ren etal. 2023), e.g. Reps nicking and joining activity for the release of circular DNA is inhibited by a plant protein, proliferative cell nuclear antigen (Bagewadi etal. 2004). If both rolling circle replication and recombinant-dependant replication occur, it is possible that the balance is shifted as comparison of DNA forms between systemic and infiltrated tissue suggest the recombination-dependant form is emphasised in the initial agroinfection (Jeske etal. 2001). Leading to the question, does agrobacterium inoculation essentially force the virus to escape from a pool of defective viral DNAs, essentially slowing true initiation of infection which in turn gives the plant silencing a leg up? Agrobacterium strains Agrobacterium is a pathogen on its own and in addition to DNA, it also transfers virulence proteins to the plant which disables the bacterial defence response and promotes transformation (Lacroix and Citovsky 2022). Notably, the agrobacterium effector 6b can interfere with the silencing machinery (Wang etal. 2011). Much like geminivirus strains have preferred hosts, so too do the strains of agrobacterium. The individual lab strains can also differ in their chromosomal background, Ti-plasmid and/or small genomic alterations such as loss or gain of a selection marker (Hellens etal. 2000; De Saeger etal. 2021), which makes optimising the methods across different research teams challenging. For example, the C58C1 (pTiB6S3ΔT)H is the C58 chromosomal background with its native Ti-plasmid replaced by the Ti-plasmid from the wild strain B6S3 where the T-DNA region has been deleted (Petit etal. 1978; Deblaere etal. 1985). Finally, the H denotes that the strain contains the helper plasmid pCH32 which increases the expression of some virulence genes (Hamilton 1997). Unfortunately, often names have been simplified in the plant community, such that C58C1(pTiB6S3ΔT)H has been written as C58C1 and GV3101(pMP90) simply as GV3101. Both these plasmids originally denoted agrobacterium strains without a Ti-plasmid and thus lacking many key genes for the transfer of DNA. In this review, we have not renamed agrobacterium strains from what they are referred to in publications. In agroinfection of tomato and zucchini plants with tomato leaf curl New Delhi virus (ToLCNDV), changing the agrobacterium strain from AGL1, EHA105 or GV3101 to LBA4404 doubled the efficiency of infection (Ruiz etal. 2017). Here, LBA4404 has the Ach5 chromosomal background whilst AGL1, EHA105 and GV3101 belong to the C58 chromosomal background. Another study with agroinfection of tomato with tomato yellow leaf curl Kanchanaburi virus (TYLCKaV) with two different strains of agrobacterium, GV2260 and EHA105, resulted in different disease incidences (Koeda etal. 2017). The Ach5 background also had higher disease incidence over C58C1 for mungbean yellow mosaic India virus (MYMIV) in mungbeans. Inoculation of DNA-A with Ach5 and DNA-B with C58C1 produced high disease incidence (Table1) (Jacob etal. 2003), indicating an effect from the agrobacterium effectors. Whilst studies indicate an effect from the choice of Agro strains on different plant species, there is no evidence to select a universally better strain. This calls for more agroinoculation studies comparing the efficiency of different strains whilst keeping all other parameters constant. This is also the case for plant transformations where different plant species are preferentially transformed with different agrobacterium strains (Chetty etal. 2013; Kassahun etal. 2021). Whilst other parallel parameters such as silencing and necrotic response to agroinfections are the major limitations for the expression of the T-DNA by the agrobacterium (Kuta and Tripathi 2005; Zipfel etal. 2006). In Nicotiana tabacum, the GV3101 agrobacterium strain induces a stronger defence response than LBA4404, as measured by reactive oxygen species and resistance to Pseudomonas syringae (Sheikh etal. 2014). This induction of plant defences inhibited tobacco mosaic virus (TMV) infection in leaf regions previously infiltrated with agrobacterium (Pruss etal. 2008). Comparison of different agrobacterium strains for the stable transformation of N. tabacum was likewise better with LBA4404 (Bakhsh etal. 2014). Inoculation designs andoptimisation It is not just the agrobacterium strain which influences geminivirus infection, the where and when of the agroinoculation also influence the disease incidence. The relative expression of relevant plant genes for infection and defence or lack thereof undoubtedly varies in place and time in the plant. Summaries of studies on these topics are listed in Table1. Various methods have been tried such as the injection of agrobacterial suspension on the abaxial surface of the leaf Plant Cell Reports (2025) 44:189 Page 7 of 22 189 Table 1 Different agroinfection experiments mentioned in this review Virus (Genbank accession) Plant—variant Inoculation method Agrobacterium strain Inoculation media Stage of plant Infectivity Reference Malvastrum leaf curl Guangdong virus (AM236779) Petunia hybrida Stem puncture EHA105 Bacterial media 4 to 6 leaf stage 100% (30) (Wu etal. 2007) Malvastrum coromandelianum 0% (?) Bean golden yellow mosaic virus (AF173555 and AF173556) Phaseolus vulgaris— susceptible cultivars Stem puncture—first internode LBA4404 2 × concentration of yeast tryptone media, OD600 of 1 10 to 12 days old plants 73% (244) (Garrido-Ramirez etal. 2000) Phaseolus vulgaris— resistant cultivars 10 to 12 days old plants 6% (112) Tomato yellow leaf curl virus (AJ519441) Solanum lycopersicum—cv. Marmande Stem puncture LBA4404 Lysogeny broth, OD likely high Five to six leaf stage 100% (10) (Morilla etal. 2005) Capsicum annuum—cv. Cadia 0% (40) Vernonia yellow vein virus (AM182232) Vernonia cinerea Stem puncture—first internode EHA105 0.9% NaCl, OD600 of 1.2 to 1.5 40 to 45 days old plants 64% (20) (Packialakshmi and Usha 2011) Leaf infiltration 40 to 45 days old plants 0% (?) Cucurbit leaf crumple virus (NC_002984 and NC_002985) Citrullus lanatus—multiple cultivars Stem puncture— beneath the shoot apex and at first internode C58 Bacterial media, OD600 of 1 2 true-leaf stage 77%-87% (Hagen etal. 2008) Cucurbita pepo—multiple cultivars 13%-84% Cucumis melo—multiple cultivars 0–60% Cucurbit leaf crumple virus (PP617367 and PP617368) Cucurbita pepo. cv. Gold Star Leaf puncture by dermal microneedle roller EHA105 MM, OD600 of 1 2 true-leaf stage 90% (20) (Kavalappara etal. 2024) Water, OD600 of 1 100% (20) Mungbean yellow mosaic India virus (KY556680 and KY556679 Vigna unguiculata—cv. Walp Leaf infiltration into young trifoliate leaves EHA105 MM, 200µM acetosyringone, pH 5.6 OD600 of 0.6 4 weeks old 100% (5) (Kumar etal. 2017) Vigna unguiculata—cv. Walp. Stable RNAi lines 24% (45) Mungbean yellow mosaic geminivirus (AY049772 and AY049771) Vigna mungo—cv. Hepper Stem puncture (Near cotyledon) C58 (TiC58) Agrobacterium resuspended in water 2 days old seedling 40% (30) (Mandal etal. 1997) Seed puncture Seeds submerged 1 day in water 13% (30) Plant Cell Reports (2025) 44:189189 Page 8 of 22 Table 1 (continued) Virus (Genbank accession) Plant—variant Inoculation method Agrobacterium strain Inoculation media Stage of plant Infectivity Reference Sweet potato leaf curl virus (JX286654.1) Nicotiana benthamiana Stem puncture GV3101 MM, 200µM acetosyringone OD600 of 1, pH 5.6 2 weeks old 80% (10) (Zhang etal. 2024) 1 day pre-incubation in AB with 200µM acetosyringone, followed by inoculation in 100% (10) MM with 200µM acetosyringone, OD600 of 1, pH 5.6 Pepper yellow leaf curl Indonesia virus (LC051114 and AB213599) Capsicum annuum—cv. No. 218 Leaf infiltration on the abaxial sides of the cotyledons GV2260 MM, 400 µM acetosyringone OD600 of 0.1 2 weeks old seedling 92% (13) (Koeda etal. 2018) MM, 400 µM acetosyringone OD600 of 1 8% (13) Maize streak virus Zea mays Stem puncture into stem and bundled sheaths EHA105 MS, pH 5.5 40 to 45 days old plants 88% (43) (Grimsley etal. 1987) Mungbean yellow mosaic India virus Vigna radiata—cv. R. Wilczek Stem puncture EHA105 Bacterial media, OD600 of 1 2 to 3 true leaves stage 98% (132) (Li etal. 2015) Mungbean yellow mosaic India virus— point mutation removing AC5 reading frame 24% (132) Maize streak virus Zea mays Stem puncture into stem and bundled sheaths EHA105 MS, pH 5.5 40 to 45 days old plants 88% (43) (Grimsley etal. 1987) Mungbean yellow mosaic India virus Vigna radiata—cv. R. Wilczek Stem puncture EHA105 Bacterial media, OD600 of 1 2 to 3 true leaves stage 98% (132) (Li etal. 2015) Mungbean yellow mosaic India virus— point mutation removing AC5 reading frame 24% (132) Plant Cell Reports (2025) 44:189 Page 9 of 22 189 Table 1 (continued) Virus (Genbank accession) Plant—variant Inoculation method Agrobacterium strain Inoculation media Stage of plant Infectivity Reference Tomato golden mosaic virus (NC_001507 and NC_001508) (single construct/agrobacterium) Nicotiana benthamiana Stem puncture at stem base and 5 cm above PC2669 (pTiC58) Yeast extract beef broth, 2 × 109 cells pr. 20µl 4 to 6 leaf stage 95% (20) (Hayes etal. 1988) Tomato golden mosaic virus (NC_001507 and NC_001508) (separate constructs/ agrobacterium) 4 to 6 leaf stage 70% (20) Yeast extract beef broth, 2 × 104 cells pr. 20 µl 4 to 6 leaf stage 40% (20) Yeast extract beef broth, 2 × 103 cells pr. 20 µl 4 to 6 leaf stage 0% (20) Tomato yellow leaf curl virus (AM282874) Solanum lycopersicum—cv. moneymaker Stem puncture EHA105 MS, 100 µM acetosyringone, OD600 of 1 4 to 6 leaf stage 48% (?) (Liu etal. 2021) Leaf infiltration 40% (?) INABS 72% (?) Tomato yellow leaf curl Kanchanaburi virus (AB921568 and LC177332) Solanum lycopersicum—cv. Momotaro 6cm of shoots are submerged in agrobacterium suspension GV2260 MM, 200µM acetosyringone, pH 5.6, OD600 of 0.6 First true-leaf stage 4% (57) (Koeda etal. 2017) EHA105 41% (45) Stem puncture with toothpick coated in agrobacterium GV2260 Raw agrobacterium 4% (73) EHA105 92% (76) Tomato yellow leaf curl virus (AB116632) Solanum lycopersicum—cv. Shugyoku 6cm of shoots are submerged in agrobacterium suspension C58C1 MM, 200 µM acetosyringone, pH 5.6, OD600 of 0.6 Fifth adult leaf fully expanded 93% (16) (Yamaguchi etal. 2013) 6cm of shoots are submerged in agrobacterium suspension and vacuum infiltrated 100% (16) Ageratum yellow vein virus + β-satellite (X74516 and AJ252072) (separate constructs/agrobacterium) Ageratum conyzoides Stem puncture GV3850 Water, not detailed 3 to 5 leaf stage 6.3% (63) (Saunders etal. 2001) Ageratum yellow vein virus + β-satellite (X74516 and AJ252072) (single construct/agrobacterium) 86% (22) Plant Cell Reports (2025) 44:189189 Page 16 of 22 explanation as to why biolistic delivery is less efficient. In a screen of different cultivars of beans for resistance to bean golden yellow mosaic virus (BGYMV), inoculation was carried out with both agrobacterium, sap rub and particle bombardment. In this experiment, agroinfection gave lower disease incidence in resistant cultivars than particle bombardment or sap rub (Garrido-Ramirez etal. 2000) indicating that one approach does not work for all virus–plant combinations and/or that optimisation of approach is more important. Generally, biolistic delivery is less common as it is more laborious and costly, requiring preparation of the individual shots for each plant. Other considerations regardinggeminivirus infectious clones N. benthamiana, owing to the ease by which it can be infected by most geminiviruses, readily provides a host for testing the geminivirus infectivity assays. However, it is resistant to insect feeding and thus difficult to use as a source plant for insect transmission (Davino etal. 2009). Recently, however, gene-edited N. benthamiana, made palatable for whiteflies by a deficiency in acylsugar, was found to sustain whiteflies for transmission (Feng etal. 2022; Thompson etal. 2024). Such plants can be used if transmission by insects is an essential part of your studies. Agroinfection methods have often sought to increase agrobacterium delivery to overcome low disease incidence. Visually tracking the initial infection would be useful in understanding the causes of inoculation escape. However, one of the limitations is the lack of easily detectable markers for virus infections. Geminiviruses select for their genome size (Etessami etal. 1989; Rojas etal. 1998), which makes it impossible to add a marker such as a GFP cassette to the virus coding sequence without compromising its virulence and function in some way. The only exception to this is a few bipartite begomoviruses that retain movement with their CP replaced with GFP, which is unnatural and makes the GFP expression dependent on initiation of viral replication (Padidam etal. 1995; Sudarshana etal. 1998; Levy and Czosnek 2003). To overcome the limitations of efficient gene expression, Agrobacterium strains have been developed with new engineered agrobacterium strains delivering additional effectors to suppress host defences. The reduction in silencing increases the expression and transformation. This has not been tested on geminivirus infection, and whether this would translate into higher disease incidence remains to be seen (Raman etal. 2022). Another upcoming technology to pay attention to is the development of nanoparticles for DNA delivery to plant cells (Cunningham etal. 2018). Plasmid DNA linked to cell penetrating peptides has been shown to enter leaves and express the β-glucuronidase reporter gene (Thagun etal. 2022). Such nanoparticles carrying the viral DNA could be injected into plants like agrobacterium. How and if they affect disease incidence also remain unknown (Zuverza-Mena etal. 2017). Whilst some virus–host combinations allow an infection to start and spread if any cell is infected, many fail to establish outside of the phloem. The right virus in the right cell can make all the difference for the development of disease. Initial infection by geminivirus has evolved around the assumption that the virus is deposited into the phloem by the feeding insects and not into the mesophyll tissue by humans. Silencing of virus replication by the plant in the mesophyll tissue might be a reason for the failed infections by the geminivirus clones. The silencing machinery also spreads in mesophyll, leading to a potential dynamic where siRNA from aborted infections silences the DNA reaching the phloem before the virus does. A better understanding of the underlying mechanisms in the initial infection would therefore also be connected to a better understanding of tissue tropism of geminiviruses. Comparison between biolistic and agrobacterium-mediated inoculations is rare, as very few studies use both approaches. Such studies would be crucial for uncovering the impact of phloem limitations in initial geminivirus infection. There is a question of whether agrobacterium delivery assists in the generation of defective interfering DNAs, incomplete replicated virus DNAs molecules which may assist in promoting RNAi (Patil and Dasgupta 2006; Bach and Jeske 2014). In addition, stable expression of VirE2 in plants attenuates symptoms and reduces begomovirus DNA accumulation (Sunitha etal. 2011; Resmi etal. 2015; Yousaf etal. 2020), possibly due to the single-stranded binding, as single-stranded DNA binding proteins interfere with begomoviruses (Rasool etal. 2016). Part of the continued research into understanding distinct virus factors found throughout the family Geminiviridae involves recombinant viruses (Li etal. 2015; Lee etal. 2020; Vo etal. 2023). A high success rate with infectious clones will aid in easier interpreting of results and reduce the workload and number of plants to be tested. Conclusions Comparison of experimental methods in the literature is challenging considering factors which majorly influence disease incidence might not have been recorded. Exactly where on the stem the plant is pricked, the exact pH of the infiltration buffer, exact conditions of the plant and more can all matter. Plant susceptibility and virus host range can come down to small variations, further complicating like-forlike comparison. It is therefore hard to suggest a universal approach to limit inoculation escape. We therefore provide Plant Cell Reports (2025) 44:189 Page 17 of 22 189 the following general guidelines: i) carry out the experiment in a single setup to limit unknowable environmental variables; ii) young seedlings of not more than 2–3weeks of age are preferable for agroinfection; iii) stem puncture is a safer bet than leaf infiltration; iv) injecting the epicotyl region or upper stem is better; v) changing agrobacterium background and/or Ti-plasmid can significantly alter disease incidence, if the initial trials do not work out; vi) optimising agrobacterium virulence gene induction is unlikely to improve disease incidence; vii) OD600 of 1 is common, but lowering it tenfold might in some cases improve disease incidence; viii) for bipartite begomoviruses, unless swapping DNA-B components is part of the plan, it could be advantageous to clone DNA-A and -B into one vector. Finally, with the variability introduced by the methods, it would be advisable to test the system on a known susceptible variety and alter the conditions if high levels of infections (> 100% or close to it) are not achieved consistently. The infectivity is the percentage of inoculated plants developing symptoms, with the total number of plants tested in the experiment noted in parenthesis, if provided; otherwise, a (?) is shown. Genbank accession of infectious clones is provided if available; otherwise, variant information is provided. For methods, see (Fig.3). Agrobacterium strains are written without Ti-plasmid if unmentioned in their publication. Bacterial media—the liquid media used to grow the bacterium was used as inoculation; this is likely yeast extract beef broth, yeast extract tryptone or lysogeny broth media, but the publication does not clarify it. OD600 optical density at 600nm, cv. cultivar. Inoculation media abbreviations, MM magnesium and MES, common inoculation buffer with 10mM MgCl2, 10mM MES and pH usually 5.5 to 5.8; AB agrobacterium minimal media (Wu and Lai 2022), MS Murashige and Skoog basal medium (Murashige and Skoog 1962). The infectivity is the percentage of inoculated plants developing symptoms, with the total number of plants tested in the experiment noted in parenthesis, if provided; otherwise, a (?) is shown. Genbank accession of infectious clones is provided if available; otherwise, variant information is provided. For methods, see (Fig.3). Agrobacterium strains are written without abbreviations: OD600—optical density at 600nm; cv.—cultivar. Conflict ofinterest The authors have no conflict of interest to declare that is relevant to the content of this article. Acknowledgements This publication has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 101000570, the project Virtigation. Author contributions SM and MG came up with the idea to review the literature of methodologies; SM did the literature search, and both the authors wrote the manuscript. Both the authors have read and approved the manuscript. Funding HORIZON EUROPE European Innovation Council, 101000570, Maruthi M.N. Gowda. Data availability Data sharing is not applicable to this article as no new datasets were generated or analysed during the current study. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. 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