CN102933713B - Viral-based transient-expression vector system that allows multiple applications - Google Patents

Abstract

Disclosed herein are viral vectors suitable for transfection into woody trees for purposes of delivering and expressing beneficial genes. Specifically exemplified herein are vectors for transfecting citrus trees. The vectors allow for the expression of useful proteins, such as those that can protect the tree from disease. Specifically exemplified herein are methods of transfecting woody trees that allow multiple applications of vectors while avoiding superinfection exclusion.

Description

Multifunctional transient expression viral vector system

Cross References to Related Applications

This application claims U.S. Patent Application No. 61/321,970, filed August 8, 2010, and U.S. Patent Application No. 61/440,445, filed February 8, 2011, under Title 35, United States Code, Section 119 (35USC 119) priority.

field of invention

The original embodiment of the invention concerned a viral-based transient expression vector that could express a foreign gene in a daughter line for an extended period of time, allowing similar vectors to be used on the same daughter line at the same time or at a later time. Other embodiments relate to viral vector structures and methods that avoid ruling out superinfection, thereby diversifying the vector's applications.

Background technique

Virus-based transient expression vectors are common tools used in plant molecular biology experiments around the world, which can rapidly express or silence genes in plants. They are also important tools in plant genomics, allowing the screening of sequence sequences for a function. However, usable vectors can only be developed from a limited number of similar viruses in herbaceous plants. Well-known examples are tobacco mosaic virus (TWV)-based vectors (Dawson et al., 1989; Donson et al., 1991; Shivprasad et al., 1999; Rabindran and Dawson, 2001). The challenges of woody plants are even more special. Even when vectors exist that can infect trees, the time required to systematically infect and analyze genes expressed in trees generally exceeds the stability of known virus-based vectors. But challenges posed by reproductive constraints and the decades it takes to improve trees have increased the need for useful virus-based vectors.

Citrus tristetravirus (CTV) is a member of a complex family of long-teloviruses with monopartite, bipartite, and tripartite genomes that are transmitted by partial insect vectors, including aphids, whitefly and whitefly Scale (Bar-Joseph et al., 1979; Dolja et al., 1994; Agranovsky, 1996; Karasev, 2000). The long curved virion (2000nm X 10-12nm) of CTV is coated by two coat proteins: the major coat protein (CP) covers approximately 97% of the virion, while the non-major coat protein (CPm) is responsible for wrapping the other end Packed into the shell. The single-stranded RNA gene of CTV is about 19.3 kb, which is divided into twelve open reading frames (ORFs) (Pappu et al., 1994; Karasev et al., 1995) (Figure 1). ORF 1a encodes a 349 kDa polyprotein that includes two papain-like protease domains and transmethylase-like and helicase-like domains. Translation of the polyprotein is thought to proceed through an a+1 frameshift in the polymerase-like region (ORF 1b). Both ORFs 1a and 1b plus the non-coding ends are required for protoplast replication (Satyanarayana et al., 1999). Ten 3' ORFs are expressed by 3' co-terminal subgene (sg) mRNAs (Hilf et al., 1995; Karasev et al., 1997). In addition to the two coat proteins, p65 (HSP70 homolog) and p61 are required for efficient virion assembly and for passage of the virus between protoplasts, thereby enriching lemon tree-infected buds (Satyanarayana et al ., 2000). The p6 protein is required for plant infection, while the p20 and p23 proteins and CP are suppressors of RNA silencing (Lu et al., 2004).

CTV can infect and move in some citrus varieties in which some viral genes have been deleted. CTV contains five genes, p6, p33, p18, p13 and p20, in the 3' portion of the genome not required for virion replication or formation. p33, p18, and p13 are not conserved among other members of this group of viruses and are thought to have evolved to ensure specific interactions with citrus hosts. We found that deletions in the p33, p18, or p13 ORFs, respectively, resulted in a modest loss of virus infection, amplification, and transmissibility among citrus trees (Tatineni et al., 2008). Moreover, deletion of any combination of p33, p18, or p13 genes allowed the virus to infect citrus trees as a whole, including deletion of all three genes. GFP-tagged CTV mutations and deletions in p33 ORF or p33, p18 and p13 ORF demonstrated that the movement and distribution of these deletion variants were similar to wild-type viruses.

In superinfection exclusion or cognate interference, preexisting infection prevents secondary infection by the same or a similar virus but cannot affect infection by an unrelated virus. This phenomenon was first discovered by McKinney (McKinney, 1926; 1929) between two genotypes of tobacco mosaic virus (TMV), and subsequently also in phages (Dulbecco, 1952; Visconti, 1953). Since then, often also in animals (Adams and Brown, 1985; Bratt and Rubin, 1968; Delwart and Panganiban, 1989; Geib et al., 2003; Johnston et al., 1974; Karpf et al., 1997; Lee et al. al., 2005; Singh et al., 1997; Steck and Rubin, 1966; Strauss and Strauss, 1994; Whitaker-Dowling et al., 1983; Wildum et al., 2006) and plants (Bennett, 1951; Fulton, 1978 ; Gal-On and Shiboleth, 2005; Hull and Plaskitt, 1970; Hull, 2002; Lecoq et al., 1991; Salaman, 1933; Walkey et al., 1992) viruses. In plant virology, homologous interference was first used as a test of viral relatedness to determine whether two virus isolates were the same "strain" of the virus or represented different viruses (McKinney, 1929; Salaman, 1933). Subsequently, the technology was developed into a management tool to reduce crop loss by using mild virus isolates to purposely infect plants to reduce infection and damage caused by more severe virus isolates, which has been termed " Cross-protection" (see Gal-On and Shiboleth, 2005 and Hull, 2002 for review).

Description of drawings

(A) in Fig. 1 is a schematic diagram of the gene organization of wild-type CTV T36 (T36CTV9R). (B) and (C) are illustrations of the structure of Δp33CTV and the hybridized structure substituted by the pro-protease domain, respectively. Blank boxes indicate ORFs and their translation products. PRO, papain-like domain; MT, methyltransferase; HEL, helicase; RdRp, an RNA-dependent RNA polymerase; HSP70h, HSP70 homologue; CPm, non-major coat protein; CP, major coat protein . The black boxes represent the alternative T68-1 sequences in the T36 genome, respectively. Arrows indicate the location of p33 ORF deletion.

Figure 2 shows a schematic representation of the hybrid virus replaced by the protease region from the T68-1 isolate (black box) into the T36 genome. Bottom: Virus multiplication in plants infected with T36 isolate or hybrid L1L2h alone (lanes 2, 3); lane 4 shows L1L2h multiplication in plants pre-infected with T36. Viral amplification was analyzed by reverse transcription-PCR reactions in which each reaction mixture contained 1 set of primers: one set specific for T36 protease and the other set targeting the protease region of T68 to differentiate T36 from L1L2h.

Detailed description of the invention

Viruses tend to prevent superinfection by related viruses. Addition of a virus-based transient vector generally prevents the use of that vector or related vectors in the same tree. The inventors also now realize that it sometimes still makes sense to add a vector to a target organism (such as a tree or plant) after the target organism is already infected with a similar vector. For example, the inventors discovered that it would be necessary to be able to add a vector if a plant lost a foreign gene being expressed; if a better gene was found to be expressed in the tree; and/or if more than one gene needed to be expressed. Likewise, the inventors have also recognized the need to apply the vector of a viral strain to a wild-type plant infected with that strain. The inventors have found that targeted modifications to the corresponding parts of the viral vector can avoid the superinfection exclusion phenomenon.

The corresponding embodiment of the invention described here is a virus structure based on citrus tristetra virus (CTV) and a positive-sense RNA long-line virus construction, which are capable of double infection in plants already infected by a virus of a similar strain. Infect. The inventors found that the viral structure constructed by modifying wild-type CTV viruses could solve the problem of superinfection exclusion, for example, by having their proprotease replaced by a proprotease sequence from a different virus species.

According to other embodiments, the present invention also relates to viral constructs based on other citrus tristetraviruses (CTV) and a positive-sense RNA long line virus, which are capable of superinfection with subsequently introduced CTV viral constructs . The inventors found that the virus structure constructed by modifying the wild-type CTV virus, such as the virus lacking the gene encoding the functional p33 protein, could not protect against the infection of the wild-type virus. When successive plant buds were used with viral constructs lacking the p33 protein and wild-type CTV, primary infection of plants with deletion mutant viral constructs had no significant effect on the establishment of secondary infection with wild-type virus. Thus, deletion of the p33 ORF produces a "non-cross-protecting" variant of CTV.

Previously, the inventors examined the relationship between the ability of different genotypes of citrus tristetravirus (CTV) to prevent superinfection by other virus isolates. Exclusion of superinfection occurred only between isolates of the same CTV strain. When using isolates of the same line for sequential plant budding, the first infection provides complete protection against the dangerous isolate. An example of complete cross-protection between CTV viruses was noted when infection with GFP-expressing CTV was completely suppressed in plants primed with wild-type CTV belonging to the same T36 line.

According to one example, the inventors examined the superinfection exclusion of viral constructs based on infectious cDNA clonal constructs of the T36 strain of CTV. The results showed that superinfection exclusion was not provided for wild-type virus infection by modifying the viral structure of the wild-type CTV virus construct, for example, these viruses lacked the gene encoding a functional p33 protein or had a proprotease region from another CTV strain (T68) substitution. When one of the virus constructs was used for primary budding of citrus trees, followed by budding with wild-type CTV, the primary infection of plants with the mutant virus had no significant effect on secondary infection with the wild-type virus. Thus, deletion of the p33 ORF and substitution of the pro-protease region produces "non-cross-protecting" variants of CTV. These virus constructs can be used as plant vectors to achieve diverse applications.

In another embodiment, the invention relates to a viral vector construct where the viral construct is engineered to replace the endogenous proprotease of the viral vector with a proprotease from an isolate of a different strain of a commonly used virus species. Strains of CTV are defined as different strains from the system according to the 1a ORF nucleotide sequence analysis of the virus (Hilf et al., 2005). Using this definition, T36 and T68 are lines. Each virus sample is referred to as an isolate of one of these lines. Each line was named a "type isolate" and consisted of isolates with minor sequence differences from members of the type. In a more specific embodiment, the viral vector is a CTV isolate in which the proprotease sequence has been replaced by an isolate of another CTV strain. In a more specific embodiment than the previous one, the CTV vector is constructed based on an isolate of the T36 strain, whose proprotease sequence is replaced by that of an isolate of the T68 CTV strain. In a more specific embodiment, the substituted protease sequence is a papain-like region.

According to another embodiment, the invention relates to a method of alleviating the exclusion of CTV viral vector superinfection by continuous viral budding. The method comprises inoculating the target plant for the first time with a CTV virus vector that has lost or destroyed the p33 ORF, so that the first CTV virus vector infects the target plant to produce an infected plant, and then allowing the infected plant to inoculate the second CTV virus A vector with or without the pss gene or a disrupted p33 gene. The second CTV viral vector is capable of infecting already infected plants. In a specific embodiment, the target plant is a citrus fruit tree. In another specific embodiment, primary and/or secondary CTV vectors are configured to include an expressible sequence encoding a heterologous protein.

According to another embodiment, the invention relates to a method of alleviating the exclusion of CTV viral vector superinfection by continuous viral budding. The method involves sequentially inoculating a target plant with a first and a second viral vector. The first viral vector was constructed so that a pro-protease sequence was replaced by a homologous pro-protease sequence from an isolate of another CTV strain. In specific embodiments, the first and second viral vectors are from a commonly used virus species. In a more specific embodiment, the first and second viral vectors comprise proprotease regions from isolates of different CTV strains. In a specific embodiment, the target organism is a plant, and in a more specific embodiment, the plant is a citrus fruit tree. In another specific embodiment, the first and/or the second CTV vector is configured to include an expressible sequence encoding a heterologous protein.

The inventors realized that sometimes after the target organism had been infected with a similar vector, the target organism

(such as trees or plants) is still meaningful. For example, the inventors discover that it would be necessary to be able to add a vector if a vector in a plant is missing a foreign gene being expressed; if a better gene is found to be expressed in trees; and/or if expression of more than one gene is desired. Likewise, the inventors have also recognized the need to apply the vector of a viral strain to a wild-type plant infected with that strain. The inventors have found that targeted modifications to the corresponding parts of the viral vector can avoid the superinfection exclusion phenomenon.

The inventors found that the virus structure constructed by modifying wild-type CTV viruses can solve the problem of superinfection exclusion, for example, by having their proprotease replaced by a proprotease sequence from a different virus species.

The relationship between different genotypes of citrus tristetravirus (CTV) in their ability to prevent superinfection by other virus isolates was examined. The results showed that superinfection exclusion only occurred between isolates of the same CTV strain. When using isolates of the same line for sequential plant budding, the first infection provides complete exclusion of at-risk isolates. An example of complete cross-protection between CTV viruses was noted when infection with GFP-expressing CTV was completely inhibited in plants primed with wild-type CTV belonging to the same T36 line (Folimonova et al., 2010).

According to one example, the inventors examined the superinfection exclusion of viral constructs based on infectious cDNA clonal constructs of the T36 strain of CTV. The results showed that by modifying the virus structure of the wild-type CTV virus construct, such as replacing the L1L2 protease region with a homologous sequence from a different virus strain, the constructed virus vector could be used even in plants that had been infected by the same virus strain. Double infection can be avoided. Accordingly, viral vector embodiments of the present invention can be used as vectors for trees that have been previously infected with a virus of the same strain, such as trees growing in the field infected by natural transmission of the virus or due to earlier use of a construct based on the same strain of virus. The tree infection caused by the CTV vector, so as to avoid the exclusion of secondary virus vector infection.

In another embodiment, the invention relates to a viral vector construct where the viral construct is engineered to replace the endogenous proprotease of the viral vector with a proprotease from an isolate of a different strain of a commonly used virus species. Strains of CTV are defined as different strains from the system according to the 1a ORF nucleotide sequence analysis of the virus (Hilf et al., 2005). Using this definition, T36 and T68 are lines. Each virus sample is referred to as an isolate of one of these lines. Each line was named a "type isolate" and consisted of isolates with minor sequence differences from members of the type. In a more specific embodiment, the viral vector is a CTV isolate in which the proprotease sequence has been replaced by an isolate of another CTV strain. In a more specific embodiment than the previous one, the CTV vector is constructed based on an isolate of the T36 strain, whose proprotease sequence is replaced by that of an isolate of the T68 CTV strain. In a more specific embodiment, the substituted protease sequence is the L1L2 region.

According to another embodiment, the invention relates to a method of alleviating the exclusion of CTV viral vector superinfection by continuous viral budding. In a specific embodiment, the method may comprise allowing the target plant to inoculate the first CTV viral vector based on the first strain of CTV, whereby the first CTV viral vector infects the target plant to produce an infected plant, This infected plant was then inoculated with a second CTV viral vector based on the same CTV strain but modified to include the proprotease sequence of a different virus strain (the second). The second CTV viral vector may also include a gene for expressing a protein that yields beneficial results. In a specific embodiment, the first line is T36 and the second line is T68. The second CTV viral vector was allowed to infect already infected plants. In a specific embodiment, the target plant is a citrus fruit tree. In another specific embodiment, the first and/or the second CTV vector is configured to include an expressible sequence encoding a heterologous protein.

According to another embodiment, the invention relates to a method of alleviating superinfection exclusion with viral vectors. The method involves inoculating a target plant with a viral vector strain that has already infected the target plant. A second viral vector was constructed in which a pro-protease sequence was replaced by a homologous pro-protease sequence from an isolate of another CTV strain. In specific embodiments, the first and second viral vectors are from a commonly used virus species. In a more specific embodiment, the first and second viral vectors comprise proprotease regions from isolates of different CTV strains. In a more specific embodiment, the protease sequence comprises a complete preprotein sequence fragment, respectively consisting of 800 base pairs (bp) or less, 700 bp or less, 600 bp or less, 500 bp or less, 400bp or less, 300bp or less, 200bp or less or 100bp or less composition. Alternatively, the vector consists of an entire proprotease sequence that is at least a 100bp, 200bp, 300bp, 400bp or 500bp fragment. A non-limiting description of examples of known proprotease sequences is discussed in the References section below. In a specific embodiment, the target organism is a plant, and in a more specific embodiment, the target organism is a tree, and in a still more specific embodiment, the target organism is a citrus tree. In another embodiment, the first/second CTV vector is constructed to include an expressible sequence encoding a heterologous protein.

A virus species as used herein is a group of viruses that have similar characteristics and can infect the same (or similar) host species. Reference to a "virus strain" indicates viruses that are grouped into one class, such as CTV or other virus species, but whose genetic sequence or some other characteristic differs from other viruses that are grouped into the same class.

Example:

Embodiment 1: CTV ΔP33 structure

The inventors examined several virus constructs, all of which contained p33ORF deletions constructed based on infectious cDNA clones of T36CTV (Tatineni et al., 2008), thus preventing superinfection of GFP-expressing CTV. The results show that these deletion variants can be amplified in trees, and can also infect most of the citrus trees as a whole (Tatineni et al., 2008). To assess the ability of a host plant primary infected with a CTV p33 deletion variant to be dual-infected with GFP-tagged CTV in the same host, small lime trees were first inoculated with the mutant virus. As a control group for this experiment, another group of plants were inoculated with wild-type CTV, see Figure 1, which shows a schematic representation of wild-type CTV (A) and p33 deletion variant structure (B). For both groups, primary infection was established by grafting virus-infected tissue onto tree trunks. Trim the upper leaves to encourage new foliage. Six weeks after budding, systemic infection of new leaves was confirmed by ELISA. The plants were then added with a second bark tissue containing CTV-BC5/GFP. When the graft has healed, trim the upper leaves again to induce new growth. After the second new leaf growth (starting at 6 weeks), superinfection of the trees with the virus was determined by observing the GFP fluorescence in the bark tissue of the newly formed part. Results Wild-type CTV completely prevented superinfection by GFP expressing virus: GFP fluorescence was not detected in plants first infected with CTV9R. Conversely, plants that were first infected with a mutant virus lacking a functional p33 protein all showed GFP fluorescence, as did trees that were not primed and only inoculated with the experimental virus CTVBC5/GFP. This indicates that deletion variants have no effect on GFP-tagged CTV infection.

Example 2: CTV structure with substituted protease region

In a similar experiment, the inventors examined hybrid virus structures in which the proprotease region (nucleotide positions 108-3040 in the CTV genome) was replaced with the corresponding region of the T68-1 genome, the remainder of the hybrid structure containing the sequence of T36 (the A schematic representation of the structure is shown in Fig. 1(C)). T68-1 represents an isolate in the CTV T68 line. Similar to the experiments described above, this construct was used for primary budding of citrus plants, followed later (after confirmation by ELISA to establish infection) with CTV-BC5/GFP. Wild-type CTVs were used to prime control trees (as in experiments described above). Six weeks after the second inoculation, superinfection of the trees with the virus was determined by observing the GFP fluorescence in the bark tissue of the nascent part. As expected, wild-type CTV completely prevented superinfection with GFP-expressing viruses: no GFP fluorescence was detected in plants first infected with CTV9R. In contrast, plants primed with a virus containing an alternative protease domain all showed GFP fluorescence, as did trees that were not primed and only budded with the experimental virus CTV BC5/GFP. This indicates that the hybrid virus has no effect on GFP-tagged CTV infection.

Example 3: Viruses with substituted protease regions can be de-excluded. Recently, it was experimentally investigated whether modification of the pro-protease region would provide the virus with the ability to de-exclude. We constructed a hybrid virus construct in which a region in the T36 cDNA clone containing the two proproteases L1 and L2 (nts position 108-3039 in the CTV genome) was replaced by the T68-1 isolate of the T68 strain The corresponding region was substituted, and the rest of the hybridized structure contained the sequence of T36 (Fig. 2). Plants already infected with T36CTV virus were revaccinated using the described virus constructs. As we demonstrated previously, following primary infection with a CTV isolate, reinfection with another isolate of the same strain can be completely ruled out. For example, infection with one isolate of the T36 strain can rule out reinfection with other isolates of the T36 strain, and infection with T36-based GFP-tagged viruses can be ruled out. Moreover, infection with the T36 isolate completely ruled out secondary infection with a hybrid virus constructed on the basis of the T36 isolate, in which the sequence of eight genes in the 3' genome portion was replaced (single gene sequence or sequence of several genes combined) into Corresponding sequences of T30 or T68 line isolates. Apparently, hybrid viruses in which the T68 pro-protease region was replaced by the T36 genome proved to have a peculiar behavior: the mutated virus was able to infect plants already infected by the parental T36 virus as a whole, and the virus accumulated to the same level as the infection Healthy plants of the virus had the same level of virus accumulation (Figure 1; compare lane 4 with other lanes of control buds).

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Also, see Foliminova et al., J Virol. (2010) 84: 1314-1325, for disclosure on making viral constructs and using same for infection in plants.

The disclosure of any reference cited herein is hereby incorporated by reference in its entirety to the extent that it does not contradict the teachings herein. It needs to be known that the examples and embodiments described here are only for the purpose of better illustration, and it is suggested that those skilled in the art can make various modifications or changes, and such modifications or changes should be within the spirit and scope of the present invention within.

Claims (2)

1. alleviate the method got rid of to the CTV virus vector double infection that target plant is relevant with continuous transfected virus, described method comprises:

Target plant is buddered first CTV virus vector, and this carrier is lost to fall or a destroyed p33 gene;

Make first CTV viral vector infection target plant, thus produce the plant infected first, and

The plant of primary infection is buddered second CTV virus vector, and this carrier contains or not containing p33 gene or the p33 gene that destroys, and second CTV virus vector infects the plant of primary infection further to produce the plant of a superinfection; And

Wherein said target plant is citrus.

2. method according to claim 1, wherein first and/or second CTV carrier are configured to the expressible nucleotide sequence comprising an encoding heterologous protein.