Among bore viruses, papaya ringspot virus PRSV, zucchini yellow mosaic virus ZYMV aphid-and cucumber mosaic virus CMV are three common aphid-and prevalent viruses, causingsevere losses i
Literature review and research obj€CfẽV€S - ch nghệ, 1
Literature review and research objectives
Aphid-borne viruses threatening the production of cucurbits worldwide
The Cucurbitaceae family encompasses several economically vital crops, including cucumber, watermelon, melon, squash, butternut, and pumpkin In 2019, global production figures revealed 100.4 megatons of watermelon, 27.5 megatons of other melons, and 22.9 megatons of pumpkins and squash However, cucurbits face significant challenges from viral diseases, which can lead to substantial yield losses, as they are vulnerable to over 90 different viruses, resulting in considerable economic impacts worldwide.
Viral diseases pose a significant threat to the sustainable cultivation of cucurbits, primarily due to potyviruses transmitted by aphids, begomoviruses spread by whiteflies, and tospoviruses carried by thrips Among these, aphid-borne viruses are particularly detrimental, leading to severe yield losses in cucurbit crops Notable examples include the papaya ringspot virus W type (PRSV W), zucchini yellow mosaic virus (ZYMV), and cucumber mosaic virus (CMV), which are prevalent and cause substantial damage to various cucurbit species worldwide.
Papaya ringspot virus (PRSV) is divided into two types: PRSV-P, which infects papaya and certain other plants, and PRSV-W, which primarily affects cucurbits and quinoa but not papaya PRSV-P was first identified in Oahu, Hawaii, in 1949 and has since spread to nearly all papaya-growing regions In contrast, PRSV-W was first reported in Sri Lanka in 1965 and later in Australia in 1978 Originally referred to as watermelon mosaic virus 1, PRSV-W poses a significant threat to various cucurbit crops.
Cucurbitaceae species and results in severe damage worldwide (Ali 2020).
PRSV, a member of the Potyvirus genus within the Potyviridae family, is one of over 230 virus species that comprise approximately 30% of all plant viruses (Petrov et al 2023) This genus includes notable viruses such as the papaya ringspot virus (PRSV) and zucchini yellow mosaic virus (ZYMV), characterized by their single-stranded, positive-sense RNA genomes featuring a poly-A tail at the 3’ end (Purcifull 1984; Tripathi et al 2008; Yeh et al 1992) Additionally, Potyviruses are non-enveloped and exhibit a flexuous, filamentous structure, measuring 11-20 nm in diameter and ranging from 650 nm to 950 nm in length (Inoue-Nagata et al 2022).
The genomic RNA of a potyvirus is translated into a large polyprotein, which is then processed by three virus-encoded proteases (P1, HC-Pro, and NIa) into smaller proteins This cleavage results in the formation of 9-11 functional proteins essential for the virus's life cycle.
The article discusses various proteins associated with viral functions, including the first protein (PI, 63 kDa), helper-component protein (HC-Pro, 52 kDa), third protein (P3, 46 kDa), and several smaller proteins such as 6K1 and 6K2 (both 6 kDa) It also highlights the cylindrical inclusion protein (CIP, 72 kDa), nuclear inclusion a protein (NIa, 48 kDa, which is further processed into 21 kDa NIa-VPg and 27 kDa NIa-Pro), nuclear inclusion b protein (NIb, 59 kDa), and the coat protein (CP, 35 kDa) These proteins play crucial roles in the viral lifecycle and pathogenesis, as noted in various studies (Chung et al 2008; Mishra et al 2019; Revers and Garcia 2015; Yeh et al.).
In the potyviral genome, a short open reading frame (ORF) is located within the P3 region of the polyprotein, which is responsible for producing the P3N—PIPO protein (Chung et al 2008) This structure is illustrated in Figure 1.
VIHQ/A VYHQ/S MDQYIN YIVGIG ty my VHHEIG VFEQ/S VFHQ/S v v Y y v
The schematic diagrams of potyvirus genomes illustrate the single-stranded RNA (ssRNA) genome, with the polyprotein open reading frame (ORF) depicted by an open box, while the mature proteolytic products are labeled accordingly The large ORF concludes with untranslated regions (UTR), and the intriguing Potyviridae protein ORF (PIPO) is represented by a smaller box.
PRSV has non-enveloped flexuous filamentous particles, 700-800 nm in length (Chiang and Yeh 1997) The virus has a single-stranded RNA (+) genome containing
The PRSV W virus, consisting of 10,326 nucleotides excluding the poly-A tail, is transmitted non-persistently by aphids (Yeh et al 1992; Gadhave et al 2019) This virus poses a significant threat to cucurbit species, which are highly susceptible to its effects (Rezende and Pacheco 1998) Infected crops experience severe damage, exhibiting symptoms such as mosaic patterns, leaf malformations, blistering, and mottled or malformed fruits (Guner et al 2008; Lecoq and others).
In Vietnam, a variety of hosts, including pumpkin, green cucurbit, bottle gourd, zucchini squash, cucumber, and loofah, have been identified as sources of PRSV W isolates (Desbiez 2012; Bateson et al 2002; Revill et al 2004) The prevalence of viruses in cucurbits shows that PRSV W is the most common, accounting for 47% of cases, while CMV follows with 27% (Revill et al 2004).
In 1987, PRSV W was first identified in sponge gourd in Taiwan (Huang et al 1987) Alongside ZYMV, CMV, and watermelon silver mottle virus (WSMoV), PRSV W has emerged as a significant threat to cucurbit crops in Taiwan (Peng et al 2011).
In a field survey conducted between 2016 and 2017, PRSV W was identified as a significant virus affecting squash in Taiwan, with over 43% of the collected samples testing positive The virus was notably prevalent in the northern and central regions of Taiwan (Chan et al 2019).
Zucchini yellow mosaic virus (ZYMV)
ZYMV, first isolated from zucchini squash in Northern Italy in 1973, has significantly impacted cucurbit crops worldwide The virus was first reported in Taiwan in 1982 and in Vietnam in 2008 During the 1980s, ZYMV emerged as the most damaging virus for cucurbit crops, particularly affecting the production of Luffa spp., bitter gourd, wax gourd, pumpkin, and bottle gourd in Taiwan between 1985 and 1986 Subsequently, ZYMV was recognized as the second most critical virus affecting squash in northern and eastern regions.
Taiwan and the third most important in the center and south of Taiwan (Chan et al.
In Vietnam, ZYMV has been identified in cucumber, pumpkin, and waxy gourd, with phylogenetic analyses revealing a high level of genetic diversity among the isolates, showing 79.4-98.9% identity (Ha et al 2008) The ZYMV population consists of three genetic groups, with isolates from groups B and C specifically found in the region (Moya-Ruiz et al 2023).
ZYMV is a significant virus affecting cucurbit crops, characterized by its flexuous particles measuring approximately 750 nm (Lisa et al 1981) The virus comprises single-stranded positive RNA genomes, around 9.6 kb in length, featuring a VPg at the 5' UTR and a poly (A) tail at the 3' UTR Transmitted non-persistently by aphids, ZYMV poses a threat to key crops such as cucumber, squash, melon, and watermelon Among viruses that infect cucurbit plants, ZYMV is recognized as the most commercially important (Derbalah et al 2022).
CMV is another crucial virus infecting plants of Cucurbitaceae Since it was first reported as a disease in 1916 (Doolittle 1916), CMV has infected approximately
Concurrent control of two aphid-borne potyviruses in cucurbits by fwo-in-one yaccine $ Ee sun Biaariane CS Men neeneneesvneesonesvenesnornerenenneesnaesensneeseerssenns 33
Concurrent control of two aphid-borne potyviruses in cucurbits by two-in-one vaccine
This chapter has been modified and published as the article of “Thi-Ngoc-Bich Tran,
Hao-Wen Cheng, Xing-Yun Xie, Joseph A J Raja, and Shyi-Dong Yeh 2023,
Published Online:19 Mar 2023 https://doi.org/10.1094/PHY TO-01-23-0019-R”
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Concurrent control of two aphid-borne potyviruses in cucurbits by two-in-one vaccine
Thi-Ngoc-Bich Tran, Hao-Wen Cheng, Xing-Yun Xie, Joseph A J Raja, and Shyi-Dong Yeh
Published Online: 19 Mar 2023 | https://doi.org/10.1094/PHYTO-01-23-0019-R
The use of attenuated viruses has become a common method for protecting crops from severe viral infections, particularly in cucurbits affected by the Papaya ringspot virus W-type (PRSV W) and zucchini yellow mosaic virus (ZYMV) This study reveals that co-infection of horn melon plants with two mild strains, PRSV P-type (PRSV P) HAS-1 and ZYMV-ZAC, offers effective protection against both PRSV P and ZYMV Additionally, researchers developed mild mutants of PRSV W through site-directed mutagenesis, resulting in a stable mutant, PRSV-WAC, which caused mild symptoms and enabled symptomless recovery in cucurbits Notably, horn melon plants pre-infected with PRSV-WAC and ZYMV-ZAC showed no interference in viral accumulation and no synergistic symptoms Furthermore, agroinfiltration assays indicated no additive effect on RNA silencing suppression when mixed HC-Pros were used While PRSV-WAC or ZYMV-ZAC alone could only antagonize severe strains of their respective viruses, their co-infection provided comprehensive protection against both PRSV and ZYMV.
W and ZYMV Similar results were reproduced in muskmelon and watermelon plants, indicating the feasibility of a two-in-one vaccine for concurrent control of PRSV W and ZYMV in cucurbits.
Keywords: Cross protection, cucurbits, plant virus vaccine, mild mutants, potyviruses,virus control.
The papaya ringspot virus (PRSV) and zucchini yellow mosaic virus (ZYMV) are the two most significant aphid-borne potyviruses impacting global cucurbit production PRSV is divided into two types: the P-type, which affects papaya and cucurbits, and the W-type, which primarily damages cucurbit crops Both PRSV and ZYMV belong to the Potyvirus genus, featuring a single-stranded RNA genome of approximately 10 kb These viruses are transmitted by aphids in a non-persistent manner and possess a single open reading frame that is processed into 10-11 protein products.
Efforts to control PRSV W and ZYMV in cucurbits through chemical vector control, agricultural practices, and crop rotation have proven ineffective (Abdalla et al 2017; Yeh and Gonsalves 1984) While resistant cultivars (Cardoso et al 2010; Provvidenti and Hampton 1992) and transgenic cucurbits resistant to ZYMV have been developed (Fuchs et al 1998; Wu et al 2010), the breeding process is limited by the availability of genetic resources for each cucurbit species Additionally, safety concerns regarding genetically modified crops hinder their approval in numerous countries.
Cross protection has been an effective strategy for controlling over 30 plant viruses for more than 50 years, utilizing a mild strain to safeguard crops from severe infections of related viruses (Pechinger et al 2019; Xu et al 2020; Ziebell and Carr 2010) This phenomenon involves multiple phases of host-virus interactions, with the primary mechanism being host RNA silencing, which is triggered by the protective virus to combat the incoming virus in a sequence-homology-dependent manner (Kung et al 2014; Lin et al 2007; Raja et al 2022; Tran et al 2022) Additionally, the protective virus offers protein-mediated resistance by sequestering essential host factors or disrupting critical functions of the invading viral proteins (Kung et al 2014; Pechinger et al 2019).
According to Ziebell and Carr (2010), protective viruses enhance the host's innate immunity by activating salicylic-acid signaling, which provides a non-specific defense mechanism against various pathogens, including viruses (Kung et al 2014; Murphy et al 2020).
A mild virus suitable for cross protection can be obtained by natural selection (Costa and Muller 1980; Lecogq et al 1991) or generated by temperature treatment
(Kosaka and Fukunishi 1993), UV irradiation (Motoyoshi and Nishiguchi 1988), and chemical induction (Kosaka and Fukunishi 1997; Yeh and Gonsalves 1984).
Alternatively, genetic manipulation of the viral silencing suppressor to knock out viral pathogenicity has become a new trend (Gal-On 2000; Kung et al 2014; Lin et al.
In 2007, the attenuated mutant PRSV HAS-1 was developed through nitrous acid induction from the severe Hawaii PRSV HA strain and has been effectively used in Hawaii and Taiwan for decades to combat severe PRSV P type infections in papaya Researchers have successfully created mild mutants of several potyviruses by manipulating the pathogenicity determinant HC-Pro, as evidenced by studies from Gal-On (2000), Kung et al (2014), Lin et al (2007), and Raja et al (2022) Notably, the ZYMV mutant ZYMV AC (ZAC), featuring double mutations at the A and C sites in HC-Pro, offers complete cross protection against severe homologous virus strains.
Cucurbits are often affected by multiple unrelated virus species, with co-infection by the PRSV W-type and ZYMV leading to significant economic losses globally Strategies such as cross protection using mild strains of viruses have been employed to manage infections like the pepino mosaic virus.
2018) Also, mild bivalent virus (a chimera of PRSV P-type (PRSV-P) HA5-1 and
The PRSV W type W-C vaccine offers effective protection against both P and W type PRSV strains (You et al 2005) Additionally, research has shown that Apple latent spherical virus (ALSV) vector vaccines can target three different tospoviruses (Taki et al 2013) However, when cucumber plants were inoculated with three mild strains of ZYMV, watermelon mosaic virus (WMV), and cucumber mosaic virus (CMV) in both greenhouse and field settings, the results indicated only partial protection, leading to less severe symptoms (Kosaka and Fukunishi 1997).
Recent research has demonstrated the synergistic effect of mild attenuated virus strains in cucumber plants (Kosaka and Fukunishi, 1997) However, to date, no method has been established that combines two mild viruses without a synergistic effect while simultaneously offering comprehensive protection against two unrelated virus species.
This study explores the use of two mild strains, PRSV P-HA5-1 and ZYMV-ZAC, to combat severe viruses PRSV P-HA and ZYMV-GFP in horn melon plants A modified Taiwan PRSV W-CI strain was developed by introducing double mutations in the HC-Pro, resulting in the creation of a mild mutant WAC The research assessed the mutant WAC for its reduced symptoms, virus accumulation dynamics, and stability, alongside its effectiveness in providing cross protection against PRSV W-CI Additionally, co-inoculation of PRSV-WAC and ZYMV-ZAC was tested as a dual vaccine strategy in horn melon, muskmelon, and watermelon plants for simultaneous defense against both severe strains.
Our study demonstrates that mild strains of PRSV P type HAS-1 and ZYMV-ZAC effectively protect horn melon from severe homologous virus strains Notably, co-infection with these mild strains offers complete dual protection against severe strains PRSV P-HA and ZYMV-GFP Additionally, co-infection with genetically modified strains PRSV-WAC and ZYMV-ZAC results in symptomless recovery without interference in virus accumulation This approach provides significant protection against severe strains PRSV W-CI and ZYMV across horn melon, muskmelon, and watermelon Our findings highlight the potential of a two-in-one vaccine utilizing mild genetically modified strains to combat dual aphid-borne virus threats in cucurbits.
Plant materials and virus sources
The plants of Chenopodium quinoa Willd., papaya (Carica papaya L var.
In a temperature-controlled greenhouse maintained at 25 ± 3°C without light supplements, various crops were cultivated, including Tainung No 2, horn melon (Cucumis metuliferus Naud.), watermelon (Citrullus lanatus cv Klondike II from Known-You Seed Co., Taiwan), muskmelon (Cucumis melo cv Mei Hua from Known-You Seed Co., Taiwan), squash (Cucurbita pepo var Zucchini), and tobacco (Nicotiana benthamiana).
Mild strains of viruses, such as nitrous acid-induced PRSV HA5-1 and mutant ZYMV AC, are known to cause symptomless infections in plants, particularly in C quinoa, without inducing local lesions These mild strains serve as protective agents against more severe viruses like PRSV P-HA and ZYMV-GFP, the latter derived from a severe isolate from sponge gourd In contrast, severe viruses such as PRSV W-CI, collected from luffa gourd in Taiwan, lead to significant symptoms including leaf deformation and mosaic patterns in cucurbits The PRSV P-HA virus from Hawaii also results in leaf distortion and mosaic effects Additionally, the severe ZYMV-GFP virus was developed from an infectious clone and causes pronounced symptoms To sustain their viability, PRSV P-HA, PRSV W-CI, and mild mutant ZAC are maintained in horn melon plants, while severe ZYMV-GFP is kept in squash plants, with mechanical transfer conducted every 3-4 weeks to ensure their propagation.
Co-infection of mild mutant PRSV HAS-1 and ZYMV-ZAC against two different severe potyviruses of PRSV P-HA and ZYMV-GFP in horn melon
A study investigated the co-infection of horn melon plants with PRSV HAS-1 and ZYMV-ZAC to assess their potential protective effects against the severe PRSV P-type Hawaii strain (PRSV P-HA).
In a controlled greenhouse environment at 25 ± 3°C, horn melon plants at the two-to-three leaf stage were subjected to mechanical inoculation with attenuated mutants HAS-1 and ZAC, either individually or in combination This was done using 1g of infected horn melon leaves, diluted in 10 ml of 0.01M potassium phosphate buffer (pH 7.2), ten days post-inoculation Control plants received only the buffer Virus infection was verified at 15 days post-inoculation (dpi) through indirect enzyme-linked immunosorbent assay (ELISA) using specific antisera for PRSV CP and ZYMV CP Subsequently, two fully expanded leaves from the infected plants were further inoculated mechanically with severe strains of PRSV P-HA and ZYMV-GFP, prepared similarly with 1g of infected leaves in 10 ml of buffer.
Development of bivalent vaccines for the concurrent control of papaya ringspot virus and cucumber mosaic virus in cucurbits
The papaya ringspot virus W type (PRSV W) and cucumber mosaic virus (CMV) are significant threats to cucurbit crops globally Previous research showed that the attenuated mutant WAC of the severe PRSV W-CI strain from Taiwan effectively protects various cucurbits, including horn melon, muskmelon, and watermelon This study developed recombinants using WAC as a backbone, incorporating CMV movement protein and varying lengths of CMV coat protein Infection assays in horn melon revealed that these recombinants induced mild symptoms and symptomless recovery, similar to WAC In Chenopodium quinoa, all recombinants caused infections without local lesions, and WAC-CP, WAC-CPn, and WAC-CPc remained stable over six generations Cross-protection tests indicated that WAC-MP offered partial protection against CMV, delaying severe symptoms by 10-15 days, while WAC-CP provided complete protection against both PRSV W-CI and CMV in horn melon Additionally, WAC-CP demonstrated significant protection against mixed infections in horn melon and muskmelon Overall, WAC-derived bivalent vaccines show promise for managing aphid-borne PRSV W and CMV in cucurbits.
Keywords: Cross protection, plant vaccine, cucurbits, mixed-virus infection, potyviruses,CUCUMOVIFUS.
Cucurbitaceae family plants, including cucumber, melon, watermelon, pumpkin, and squash, are vital to the economy due to their significant agricultural output Globally, the annual production of these crops reaches approximately 27.5 megatonnes of melons, 100.4 megatonnes of watermelon, and 22.9 megatonnes of pumpkins and squash, highlighting their importance in the agricultural sector.
Over 50% of plant diseases affecting cucurbits are attributed to viruses, particularly those transmitted by aphids, leading to significant damage across various cucurbit species Notably, the papaya ringspot virus (PRSV) and cucumber mosaic virus (CMV) are prevalent aphid-borne viruses that cause substantial losses in cucurbit crops worldwide.
PRSV, a unique species within the Potyvirus genus, is categorized into two types: the P type virus (PRSV P), which infects papaya, and the W type virus (PRSV W), which does not Initially identified in Australia in 1978, PRSV W poses a threat to all cucurbit species, highlighting its significant impact on agriculture.
The virus, as noted by Rezende and Pacheco (1998), leads to symptoms such as mosaic patterns, leaf malformations, blistering, and fruit deformities (Guner and Wehner 2008; Lecoq and Desbiez 2012) Notably, the characteristics of PRSV W and PRSV P exhibit significant similarities, particularly in their serological profiles.
PRSV W is a non-enveloped virus characterized by flexuous filamentous particles measuring approximately 780-800 x 12 nm It possesses a single-stranded positive-sense RNA genome of about 10,000 nucleotides, which encodes a polyprotein that is cleaved into ten functional products by three viral proteases.
CMV is another crucial virus infecting cucurbits and belongs to the genus
Cucumovirus, a member of the Bromoviridae family, was first identified as a plant disease in 1916 Since then, it has infected around 1,200 plant species across 100 different families The virus is primarily transmitted by aphids through a nonpersistent mechanism.
Cucumber mosaic virus (CMV) poses a severe threat to melon and vegetable crops, causing yield losses ranging from 30% to 100% (Tatineni and Hein 2022) Infected plants exhibit symptoms such as leaf mosaic patterns, stunted growth, distortions in leaves and fruits, and potential death (Scholthof et al 2011; Wang et al 2020) CMV consists of 29 nm icosahedral particles that contain a tripartite genome made up of single-stranded positive-sense RNA (Scholthof et al 2011) Specifically, RNA1 encodes the replicase protein, while RNA2 encodes the 2a replicase protein and the 2b protein, which acts as a silencing suppressor.
RNA3 encodes 3a movement protein (MP) and coat protein (CP) (Zhang et al 2017).
The movement protein (MP) of Cucumber Mosaic Virus (CMV) is essential for the virus's propagation within the host by binding to its viral genome Research indicates that the removal of the C-terminal 33 amino acids from CMV-MP significantly reduces its RNA binding ability (Kim et al 2004; Saray et al 2021) Furthermore, CMV-MP also interacts with the coat protein (CP) of CMV, specifically involving the C-terminal two-thirds of the CP.
The movement protein (MP) of the Cucumber Mosaic Virus (CMV) requires 29 amino acids (aa) for effective cell-to-cell movement, as demonstrated by Salanki et al (2004) Additionally, the interaction between amino acid 51 of the CMV-MP and amino acid 129 of the coat protein (CP) significantly influences symptom expression, according to Takeshita et al (2001).
The CP of CMV is a crucial multi-functional protein that plays a significant role in viral infection, influencing symptom expression, virus movement, vector transmission, and host range (Mochizuki and Ohki 2011; Saray et al 2021) Research indicates that alterations in six amino acids within the capsid protein loop (positions 191-197) can enhance CMV symptoms while impairing aphid transmission Additionally, the amino acid at residue 129 is vital for the CMV phenotype, and the 148th amino acid position in the CP is key to preventing the recovery phenomenon.
Several strategies have been implemented to control aphid-borne PRSV W and
CMV, including weed removal, agricultural practices, crop rotation, and chemical control of vectors However, aphids are the most common vectors of plant viruses (Hooks and Fereres
Cucumber Mosaic Virus (CMV) and Papaya Ringspot Virus (PRSV W) can rapidly spread through various aphid vectors, complicating control efforts due to their extensive host range Recent advancements include the development of disease-resistant cultivars and transgenic cucurbits specifically designed to combat CMV, enhancing resistance strategies in cucurbit cultivation.
Research has identified a single recessive gene, prv, that provides resistance to the Papaya Ringspot Virus-W (PRSV-W) (Guner and Wehner, 2008) By utilizing Cas9/small guiding RNA (sgRNA) technology to target and disrupt the eukaryotic translation initiation factor 4E (eIF4E) gene, scientists have successfully enhanced cucumber's resistance to potyviruses, specifically PRSV-W and Zucchini Yellow Mosaic Virus (ZYMV) (Chandrasekaran et al.).
2016) However, the breeding process relies on available genetic sources of each cucurbit species, and safety concerns about genetically modified crops hamper their approval in many countries.
For over fifty years, cross-protection has been utilized as an effective method to control plant viruses, where a mild strain is employed to shield crops from more severe related virus strains (McKinney 1929; Pechinger et al 2019; Ziebell and Carr).
2010), including CMV (Dodds 1982; Ziebell et al 2007), PRSV P (Yeh and Gonsalves
Research has shown that cross-protection against severe strains of viruses can be achieved using mild chimeric viruses A bivalent chimeric virus, created from the mild strain PRSV P HAS-1 and incorporating the coat protein of the severe strain PRSV W, effectively protects against both PRSV P and W (You et al 2005) Additionally, in Spain, the management of pepino mosaic virus (PepMV) has successfully utilized co-infection of two mild strains to provide cross-protection against severe strains of the same species (Agiiero et al.).