LAUBER EMMANUELLE (FR)
CENTRE NAT RECH SCIENT (FR)
| WO2003089647A1 | 2003-10-30 | |||
| WO2005085417A2 | 2005-09-15 |
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| CLAIMS 1. Xanthomonas microorganism wherein at least five genes encoding type III effector proteins secreted by the type III secretion system are inactivated such that the microorganism has a score equal or inferior to 1 , preferably inferior to 1 on a disease index scale, wherein the disease index has a scale of 0 to 4; and wherein the microorganism comprises a functional type III secretion system. 2. Xanthomonas microorganism of claim 1 , wherein the microorganism has a score of 0 on the disease index scale. 3. Xanthomonas microorganism of claim 1 or 2, wherein the at least five genes are inactivated by a deletion of all or part of the genes. 4. Xanthomonas microorganism of any one of claims 1 to 3, wherein the at least five genes encode type III effector proteins selected from the group consisting of AvrBsI , AvrBs2, AvrBs3, XopB, XopC1 , XopC2, XopD, XopE1 , XopE2, XopE3, XopE4, XopE5, XopF1 , XopFZ, XopF3, XopG1 , XopG2, XopG3, XopH1 , XopH2, XopH2, XopH , Xopl2, XopJ1 , XopJ2, XopJ3, XopJ4, XopJ5, XopJ6, XopK, XopL, XopM, XopN, XopO, XopP, XopQ, XopR, XopS, XopT, XopU, XopV, XopW, XopX, XopY, XopZ1 , XopZ2, XopAA, XopAB, XopAC, XopAD, XopAE, XopAFI , XopAFZ, XopAG, XopAH, XopAI, XopAJ, XopAK, XopALI , XopALZ, XopAM, XopAN, XopAO, XopAP, XopAQ, XopAR, XopAS, XopAT, XopAU, XopAV, XopAW, XopAX, XopAY, XopAZ, XopBA, XopA, HpaA, HrpW, AvrXccAI , and AvrXccAZ. 5. Xanthomonas microorganism of any one of claims 1 to 4, wherein at least 10, 15, 20, 25, 30, 35, or 40 genes encoding type III effector proteins are inactivated. 6. Xanthomonas microorganism of any one of claims 1 to 5, wherein said Xanthomonas microorganism is Xanthomonas campestris, preferably Xanthomonas campestris pv campest ris. 7. Xanthomonas microorganism of any one of claims 1 to 6, wherein the genes coding for the type III effector proteins AvrBsI , AvrXccAI , AvrXccAZ, HrpW, XopAC, XopAG, XopAH, XopAH , XopALZ, XopAM, XopAN, XopD, XopEZ, XopF, XopG, XopH, XopJ5, XopK, XopL, XopN, XopP, XopQ, XopR, XopX1 , XopXZ, and XopZ are inactivated. Xanthomonas microorganism of any one of claims 1 to 7, wherein said microorganism was deposited at the CNCM under registration number I-5530 on July 1 , 2020. Xanthomonas microorganism of any one of claims 1 to 8, wherein said microorganism further comprises at least one heterologous gene encoding a type III effector protein, or a variant or fragment thereof. Use of the Xanthomonas microorganism of claim 9 to evaluate the recognition of at least one type III effector protein by a plant. Use of the Xanthomonas microorganism of claim 10 to evaluate the tolerance or resistance of a plant to said Xanthomonas microorganism when the type III effector protein is recognized by the plant. Use of claim 10 or 11 , wherein said plant is a monocotyledonous or dicotyledonous plant, preferably a pepper, tomato, brassica, rice, wheat, barley, citrus, banana, cassava, peanut, cotton, lettuce, walnut, strawberry or bean plant. Method for the evaluation of the recognition of a type III effector protein by a plant comprising the steps of: a) introducing a heterologous gene encoding the type III effector protein into the Xanthomonas microorganism of any one of claims 1 to 8, b) inoculating a plant with an appropriate amount of the Xanthomonas microorganism obtained in step a), and c) evaluating the recognition of the type III effector protein by the plant. Method of claim 13, further comprising a step of: d) evaluating the tolerance or resistance of the plant to the Xanthomonas microorganism when the type III effector protein is recognized by the plant. Method of claim 13 or 14, wherein said plant is a monocotyledonous or dicotyledonous plant, preferably a pepper, tomato, brassica, rice, wheat, barley, citrus, banana, cassava, peanut, cotton, lettuce, walnut, strawberry or bean plant. |
Field of the invention
The present invention relates to a genetically modified Xanthomonas microorganism and to a method for evaluating plant recognition of effector proteins using said microorganism.
Background of the invention
The Xanthomonas genus encompasses a wide variety of gram-negative plant- associated bacteria, many of which are known to cause disease. Indeed, Xanthomonas is known to infect at least 124 monocot species and 268 dicot species (Leyns et al., 1984). Despite the large number of plants that are susceptible to infection, host specificity of a given Xanthomonas species is generally limited to only one or several closely related plant genera or species. Pathovars at a subspecies level have been further defined to group Xanthomonas strains causing specific disease symptoms on specific plant genera or species.
Xanthomonas infection is dependent on the secretion of as many as 40 or more effectors (referred to herein as “T3Es”) via a type three secretion system (T3SS), causing symptoms such as spots, blight, cankers, tissue rot, and/or hormone imbalance, all of which negatively impact plants (White et al., 2009). As an example, Xanthomonas campestris pv. campestris (Xcc) is responsible for black rot of the entire Brassicaceae family, including economically important vegetable crops, while Xanthomonas oryzae pv. oryzae is responsible for bacterial leaf blight in rice, considered to be the most destructive rice disease in Asia and which has been reported to reduce yield by up to 80%. Thus, further characterization of Xanthomonas T3Es, as well as the identification of plant varieties that are tolerant or even resistant to Xanthomonas-related disease, represents a major goal for the seed industry and for agriculture in general, in the hope of improving crop yield and/or reducing spoilage after harvest.
To date, the study of various T3Es within a Xanthomonas background has generally been limited to the evaluation of no more than three effectors of a given pathovar using multiple deletion mutants (see e.g., Kay et al., 2005). Deletion of multiple genes is complicated by the fact that T3Es are generally interspersed throughout the Xanthomonas genome, meaning that most genes must be removed individually. Thus, larger scale studies evaluating the effect of one or more Xanthomonas T3Es are generally performed with A robacterium tumefasciens using a method known as agroinfiltration. However, in notable contrast to Xanthomonas, Agrobacterium does not express a T3SS permitting translocation of the protein effectors themselves. Instead, Xanthomonas T3Es are transiently expressed following T-DNA transfer of the corresponding genes into plants (see e.g., Adlung et al., 2016). Some plant species are also recalcitrant to agroinfiltration, which is necessary for efficient T3E expression, or are not sufficiently hardy to withstand this treatment. Indeed, the inventors have more specifically found that Brassica species are recalcitrant to agroinfiltration (unpublished data); this approach therefore cannot be used. Agroinfiltration technique is also temperature dependent, with temperatures above 29 °C causing certain proteins involved in T-DNA transfer to become non-functional (Fullner and Nester, 1996).
While natural strains of Xanthomonas having up to four insertion sequences (IS) in T3Es have been described (see e.g., Hajri et al., 2009; Guy et al., 2013), such strains remain pathogenic (see e.g., results obtained with Xanthomonas campestris pv. campestris str. CN07 in Figure 2 of Guy et al., 2013, for which a disease index superior to 2 is observed). In particular, there is no systematic indication as to the function of each of the T3Es which comprise an IS or even the effect of each IS on the corresponding T3E in which it is present. Indeed, while in some cases the presence of an IS may be associated with a loss of function, in other cases, an IS may be associated with a gain of function or no effect. Thus, a Xanthomonas strain having up to four ISs is also not appropriate for the characterization of T3Es.
Thus, there remains a need for improved microorganisms and methods that may be used to characterize T3Es, as well as to evaluate recognition of T3E proteins as well as tolerance and/or resistance to Xanthomonas.
Brief description of the invention
The present invention addresses the above needs, providing a Xanthomonas microorganism which may advantageously be used for the study of any combination of one or more T3Es. It is also highly advantageous as it is not specific for any particular plant species or variety, in contrast to native Xanthomonas pathovars. The microorganism can thus be used to study the recognition of Xanthomonas T3E proteins in any plant species and the associated tolerance, resistance, etc., to any given T3E or combination thereof. To this end, a Xanthomonas microorganism wherein at least five genes encoding type III effector proteins secreted by the type III secretion system are inactivated such that the microorganism has a score inferior to 2, preferably equal or inferior to 1 , more preferably inferior to 1 on a disease index scale, and wherein the microorganism comprises a functional type III secretion system, is provided herein.
Preferably, the Xanthomonas microorganism has a score of 0 on a disease index scale. This microorganism represents a highly advantageous biological tool, as, in addition to the lack of host-selectivity, it provides a cheap, robust, easy, and non-destructive means of evaluating recognition of T3Es, and plant tolerance and/or resistance thereto. The T3Es furthermore require no particular modification when evaluated in the context of such a microorganism and are introduced into the target plant as such, given that the microorganism comprises a functional T3SS.
Preferably, the at least five genes are inactivated by a deletion of all or part of the genes.
Preferably, the at least five genes encode type III effector proteins selected from the group consisting of AvrBsI , AvrBs2, AvrBs3, XopB, XopC1 , XopC2, XopD, XopE1 , XopE2, XopE3, XopE4, XopE5, XopF1 , XopF2, XopF3, XopG1 , XopG2, XopG3, XopH1 , XopH2, XopH2, XopH , Xopl2, XopJ1 , XopJ2, XopJ3, XopJ4, XopJ5, XopJ6, XopK, XopL, XopM, XopN, XopO, XopP, XopQ, XopR, XopS, XopT, XopU, XopV, XopW, XopX, XopY, XopZ1 , XopZ2, XopAA, XopAB, XopAC, XopAD, XopAE, XopAFI , XopAFZ, XopAG, XopAH, XopAI, XopAJ, XopAK, XopALI , XopALZ, XopAM, XopAN, XopAO, XopAP, XopAQ, XopAR, XopAS, XopAT, XopAU, XopAV, XopAW, XopAX, XopAY, XopAZ, XopBA, XopA, HpaA, HrpW, AvrXccAI , and AvrXccAZ.
Preferably, at least 10, 15, 20, 25, 30, 35, or 40 genes encoding type III effector proteins are inactivated.
Preferably, the Xanthomonas microorganism is Xanthomonas campestris, preferably Xanthomonas campestris pv campestris.
Preferably, the genes encoding the type III effector proteins AvrBsI , AvrXccAI , AvrXccA2, HrpW, XopAC, XopAG, XopAH, XopAH , XopAL2, XopAM, XopAN, XopD, XopE2, XopF, XopG, XopH, XopJ5, XopK, XopL, XopN, XopP, XopQ, XopR, XopX1 , XopX2, and XopZ are inactivated.
Preferably, the Xanthomonas microorganism is that deposited at the CNCM under registration number I-5530 on July 1 , 2020.
Preferably, the Xanthomonas microorganism further comprises at least one heterologous gene encoding a type III effector protein or a variant or fragment thereof.
In a particular aspect, the invention relates to a use of the Xanthomonas microorganism comprising at least one heterologous gene as provided herein in evaluating the recognition of at least one type III effector protein by a plant. The invention further relates to a use of the Xanthomonas microorganism comprising at least one heterologous gene as provided herein in evaluating the tolerance or resistance of a plant to said Xanthomonas microorganism when the type III effector protein is recognized by the plant.
The invention further relates to a method for the evaluation of the recognition of a type III effector protein by a plant comprising the steps of: a) introducing a heterologous gene encoding the type III effector protein into the Xanthomonas microorganism as provided herein, b) inoculating a plant with an appropriate amount of the Xanthomonas microorganism obtained in step a), and c) evaluating the recognition of the type III effector protein by the plant.
Preferably, the method further comprises a step of: d) evaluating the tolerance or resistance of the plant to the Xanthomonas microorganism when the type III effector protein is recognized by the plant.
Preferably, the plant is a monocotyledonous or dicotyledonous plant, more preferably a pepper, tomato, brassica, rice, wheat, barley, citrus, banana, cassava, peanut, cotton, lettuce, walnut, strawberry, or bean plant.
Detailed description of the invention
Before describing the present invention in detail, it is to be understood that the invention is not limited to particularly exemplified microorganisms and/or methods and may, of course, vary. Indeed, various modifications, substitutions, omissions, and changes may be made without departing from the scope of the invention. It shall also be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Furthermore, the practice of the present invention employs, unless otherwise indicated, conventional microbiological and molecular biological techniques that are within the skill of the art. Such techniques are well-known to the skilled person, and are fully explained in the literature.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as are commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention, preferred material and methods are provided.
It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the,” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a gene” includes a plurality of genes, and so forth.
The terms “comprise,” “contain,” and “include” and variations thereof such as “comprising” are used herein in an inclusive sense, i.e., to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.
A first aspect of the invention concerns a Xanthomonas microorganism wherein at least five genes encoding type III effector proteins secreted by the type III secretion system are inactivated to such an extent that the microorganism has a score inferior to 2, preferably equal or inferior to 1 , more preferably inferior to 1 , even more preferably has a score of 0 on a disease index scale, and wherein the microorganism comprises a functional type III secretion system.
Reference to the “Xanthomonas” microorganism as used herein, refers to any microorganism belonging to the Xanthomonas genus. Said Xanthomonas microorganism is a recombinant microorganism. That is to say said Xanthomonas microorganism is a microorganism or a strain of microorganism that has been genetically modified or genetically engineered. This means, according to the usual meaning of these terms, that the microorganism of the invention is not found in nature and is genetically modified as compared to the “parental” microorganism from which it is derived. The “parental” microorganism may occur in nature (i.e., a wild-type microorganism) or may have been previously modified. The recombinant microorganism of the invention is notably modified by the deletion and/or modification of genetic elements. The microorganism may be further modified by the introduction of genetic elements. Such modifications can be performed by genetic engineering, for example by homologous recombination.
As a non-limiting example said Xanthomonas microorganism may be selected from among X. alfalfae, X. ampelina, X. arboricola, X. axonopodis, X. boreopolis, X. badrii, X. begoniae, X. bromi, X. campestris, X. cassavae, X. citri, X. codiaei, X. cucurbitae, X. cyanopsidis, X. cynarae, X. eleusinae, X. euvesicatoria, X. fastidiosa, X. frageriae, X. gardneri, X. holcicola, X. hortorum, X. hyacinthi, X. maliensis, X. malvacearum, X. maltophila, X. manihotis, X. melonis, X. oryzae, X. papavericola, X. perforans, X. phaseoli, X. pisi , X. populi, X. sacchari, X. theicola, X. translucens, X. vasicola, and X. vesicatoria. As a non-limiting example, said Xanthomonas may be a pathovar or a strain of any of the above-described pathogenic microorganisms. As an example, the pathovar is a pathovar (pv) of X. campestris, such as aberrans, campestris, raphani, armoraciae, incanae, or translucens, a pathovar of X. arboricola such as pruni, a pathovar of X. oryzae such as oryzae or oryzicola, a pathovar of X. citri such as pv citri, malvacearum, mangiferaeindicae, or anacardii, etc.
Thus, according to a preferred embodiment, said Xanthomonas is selected from the group consisting of X. alfalfae, X. ampelina, X. arboricola, X. axonopodis, X. boreopolis, X. badrii, X. begoniae, X. bromi, X. campestris, X. cassavae, X. citri, X. codiaei, X. cucurbitae, X. cyanopsidis, X. cynarae, X. eleusinae, X. euvesicatoria, X. fastidiosa, X. frageriae, X. gardneri, X. holcicola, X. hortorum, X. hyacinthi, X. maliensis, X. malvacearum, X. maltophila, X. manihotis, X. melonis, X. oryzae, X. papavericola, X. perforans, X. phaseoli, X. pisi , X. populi, X. sacchari, X. theicola, X. translucens, X. vasicola, and X. vesicatoria. Preferably, said Xanthomonas is X. campestris. Preferably, said Xanthomonas is a pathovar or a strain of any of the above-mentioned pathogenic Xanthomonas microorganisms. Preferably, said Xanthomonas is a pathovar of X. campestris, more preferably selected among pathovars aberrans, campestris, raphani, armoraciae, incanae, and translucens, a pathovar of X. arboricola, more preferably selected among pathovars corylina, juglandis, populi, poinsettiicola, celebensis, and fragariae, a pathovar of X. oryzae, more preferably selected among pathovars oryzae and oryzicola, a pathovar of X. citri, more preferably selected among pathovars citri, malvacearum, mangiferaeindicae, or anacardii. According to a particularly preferred embodiment, said Xanthomonas is X. campestris pv campestris.
By “type III effector protein,” “type three effector protein,” or “T3E” is meant herein any protein or polypeptide that is able to be injected into a plant cell via a type three secretion system. As a non-limiting example, a T3E may modulate plant cellular pathways or processes to the benefit of Xanthomonas, for example by suppressing innate immunity of the plant, interfering with cytoskeleton formation, gene expression, or vesicle transport, and/or by promoting bacterial multiplication. The T3E may be any known or putative T3E produced by Xanthomonas. As a non-limiting example, a type III effector protein may be selected from the group consisting of: AvrBsI , AvrBs2, AvrBs3, XopB, XopC1 , XopC2, XopD, XopE1 , XopEZ, XopE3, XopE4, XopE5, XopF1 , XopF2, XopF3, XopG1 , XopG2, XopG3, XopH1 , XopHZ, XopHZ, Xopll , Xopl2, XopJ1 , XopJ2, XopJ3, XopJ4, XopJ5, XopJ6, XopK, XopL, XopM, XopN, XopO, XopP, XopQ, XopR, XopS, XopT, Xopll, XopV, XopW, XopX, XopY, XopZ1 , XopZZ, XopAA, XopAB, XopAC, XopAD, XopAE, XopAFI , XopAFZ, XopAG, XopAH, XopAI, XopAJ, XopAK, XopAH , XopALZ, XopAM, XopAN, XopAO, XopAP, XopAQ, XopAR, XopAS, XopAT, XopAU, XopAV, XopAW, XopAX, XopAY, XopAZ, XopBA, XopA, HpaA, HrpW, AvrXccAI , AvrXccAZ, any variant thereof, and fragment thereof.
A “fragment” of a T3E, as used herein, refers to a part of the amino acid sequence of a T3E comprising at least all of the regions essential for its injection by a T3SS into a plant cell. Said fragment has furthermore preferably maintained any effector function which was present in the complete T3E (e.g., selected from among those listed above). The fragment may in some cases have an amino-terminal and/or a carboxy-terminal deletion compared to the corresponding full length T3E. The fragment may be of any length so long as it may still be subject to injection by a T3SS into a plant cell. Preferably, the fragment comprises the first 50 amino acids of the N-terminal encompassing predicted secretion-translocation signals. Preferably, the length of the fragment corresponds to at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, or 99% of the corresponding full length T3E.
A “variant” as used herein refers to a protein that is structurally different from the amino acid sequence of a protein of reference but that generally retains all the essential functional characteristics of said protein of reference. A variant of a protein may be a naturally-occurring variant or a non-naturally occurring variant. Such non-naturally occurring variants of the reference protein can be made, for example, by using mutagenesis techniques on the nucleic acids or genes encoding the reference protein, for example by random mutagenesis or site-directed mutagenesis.
Structural differences may be limited in such a way that the amino acid sequence of the reference protein and the amino acid sequence of the variant may be similar overall, and identical in many regions. Structural differences may result from conservative or nonconservative amino acid substitutions, deletions and/or additions between the amino acid sequence of the reference protein and the variant. As a non-limiting example, a variant may differ from a reference protein by only a single amino acid substitution. The only proviso is that, even if some amino acids are substituted, deleted and/or added, the biological activity of the amino acid sequence of the reference protein is retained by the variant.
A “variant” of a T3E as described herein includes, but is not limited to, proteins or polypeptides having amino acid sequences which are at least 60% identical after alignment to the amino acid sequence encoding an T3E as provided herein and which has a biological function that is substantially the same or at least equal to the amino acid sequence encoding an T3E as provided herein. According to the present invention, such a variant preferably has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to one of the T3Es described herein. Said functional variant furthermore has the same function as the protein provided herein. As a non-limiting example, a functional variant AvrBsI of SEQ ID NO: 12 has at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to said sequence. As a non-limiting example, means of determining sequence similarity are further provided below.
A degree of sequence identity between proteins is a function of the number of identical amino acid residues at positions shared by the sequences of said proteins. Similarly, a degree of sequence identity between nucleic acids (e.g., genes) is a function of the number of identical nucleotides found at positions shared by the sequences of said nucleic acids. The term “sequence identity” or “identity” as used herein in the context of two nucleotide or amino acid sequences more particularly refers to the residues in the two sequences that are the identical when aligned for maximum correspondence. When percentage of sequence identity is used in reference to amino acid sequences, it is recognized that positions at which amino acids are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues having similar chemical properties (e.g., charge or hydrophobicity). When sequences differ due to conservative substitutions, percent identity between sequences may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Thus, the degree of sequence similarity between polypeptides is a function of the number of similar amino acid residues at positions shared by the sequences of said proteins. The means of identifying similar sequences and their percent similarity or their percent identities are well-known to the person skilled in the art, and include in particular the BLAST programs, which can be used from the website http://www.ncbi.nlm.nih.gov/BLAST/ with the default parameters provided on that website. The sequences obtained can be further exploited (e.g., aligned) using, for example, the programs CLUSTALW (http://www.ebi.ac.uk/clustalw/) or MULTALIN (http://prodes.toulouse.inra.fr/multalin/cgi-bin/multalin.pl ), with the default parameters provided on those websites.
Specifically, identity between amino acid sequences can be determined by comparing a position in each of the sequences which may be aligned for the purposes of comparison. When a position in the compared sequences is occupied by the same amino acid then the sequences are identical at that position.
Percent identity as referred to herein is determined after optimal alignment of the sequences to be compared, which may therefore comprise one or more insertions, deletions, truncations and/or substitutions. This percent identity may be calculated by any sequence analysis method known to the person skilled in the art. Percent identity may be determined after global alignment of the sequences to be compared taken in their entirety and over their entire length. In addition to manual comparison, it is possible to determine global alignment using the algorithm of Needleman and Wunsch (1970). Optimal alignment of sequences may preferably be conducted by the global alignment algorithm of Needleman and Wunsch (1970), by computerized implementations of this algorithm (such as CLUSTAL W) or by visual inspection.
For nucleotide sequences, the sequence comparison may be performed using any software well-known to a person skilled in the art, such as the Needle software. The parameters used may notably be the following: “Gap open” equal to 10.0, “Gap extend” equal to 0.5, and the EDNAFULL matrix (NCBI EMBOSS Version NUC4.4). For amino acid sequences, the sequence comparison may be performed using any software well-known to a person skilled in the art, such as the Needle software. The parameters used may notably be the following: “Gap open” equal to 5.0, “Gap extend” equal to 0.5, and the BLOSUM62 matrix.
Preferably, percent identity as defined herein is determined via the global alignment of sequences compared over their entire length.
As a particular example, to determine the percent of identity between two amino acid sequences, the sequences are aligned for optimal comparison. For example, gaps can be introduced in the sequence of a first amino acid sequence for optimal alignment with the second amino acid sequence. The amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, the molecules are identical at that position.
The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences. Hence % identity = number of identical positions / total number of overlapping positions x 100.
In other words, the percentage of sequence identity is calculated by comparing two optimally aligned sequences, determining the number of positions at which the identical amino acid occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions and multiplying the result by 100 to yield the percentage of sequence identity.
Preferably, the Xanthomonas microorganism comprises the inactivation of at least five genes encoding for T3Es.
By “inactivation” is meant herein that a gene of interest is not expressed at all or is not expressed in the form of a functional protein. Gene inactivation may be performed by introducing mutations into the coding sequence of a gene or into non-coding sequences. Such mutations may be synonymous (i.e. , when no modification in the corresponding amino acid occurs) or non-synonymous (i.e., when the corresponding amino acid is altered). Synonymous mutations do not have any impact on the function of a translated protein itself, but may impact the regulation of the corresponding gene in such a way that the gene of interest is not expressed (i.e., if the mutated sequence is located in a binding site for a regulatory factor). Non-synonymous mutations may impact the function of the translated protein as well as its regulation, depending the nature of the mutated sequence.
In particular, mutations in non-coding sequences may be located upstream of the coding sequence (i.e., in the promoter region, in an enhancer, silencer, or insulator region, in a specific transcription factor binding site) or downstream of the coding sequence. Mutations introduced in the promoter region may be in the core promoter, proximal promoter or distal promoter. Mutations may be introduced by site-directed mutagenesis using, e.g., polymerase chain reaction (PCR), by random mutagenesis techniques e.g., via mutagenic agents (UV rays or chemical agents such as nitrosoguanidine (NTG) or ethylmethanesulfonate (EMS)), DNA shuffling, or error-prone PCR. The insertion of one or more supplementary nucleotide(s) in the region located upstream of a gene can notably modulate gene expression in such a way that the gene is inactivated.
As a further non-limiting example, a gene may be inactivated by a partial or complete deletion of the coding sequence. Such deletions may notably be introduced by homologous recombination.
Preferably, the Xanthomonas microorganism provided herein comprises the partial or complete gene deletion of at least five genes encoding T3Es. More preferably, the Xanthomonas microorganism provided herein comprises the complete deletion of at least five genes encoding T3Es.
As a non-limiting example, the Xanthomonas microorganism may comprise the inactivation of more than five genes encoding T3Es, i.e., 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, or more genes encoding T3Es.
Thus, according to a preferred embodiment, the Xanthomonas microorganism provided herein comprises an inactivation of 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, or more genes encoding T3Es. More preferably, the Xanthomonas microorganism provided herein comprises an inactivation of at least 10, 15, 20, 25, 30, 35, or 40 genes encoding T3Es. The number of genes coding for T3Es that are inactivated will of course be limited by the number of genes present in the T3E repertoire of the corresponding Xanthomonas microorganism. Preferably, the Xanthomonas microorganism comprises an inactivation of all known genes encoding T3Es.
Preferably, at least 19% of the T3Es present in the Xanthomonas microorganism are inactivated. Thus, as a particular example, in a Xanthomonas microorganism comprising 26 T3Es, at least 5 T3Es are inactivated. More preferably, at least 20%, 25%, 30% or 35% of the T3Es present in the Xanthomonas microorganism are inactivated. Even more preferably, at least 40% of the T3Es present in the Xanthomonas microorganism are inactivated. As a particular example, in a Xanthomonas microorganism comprising 26 T3Es, at least 11 T3Es are inactivated. As an alternative example, in a Xanthomonas microorganism having 13 T3Es, at least 6 T3Es are inactivated. More preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of the T3Es present in the Xanthomonas microorganism are inactivated. When the Xanthomonas microorganism comprises the inactivation of at least five genes encoding T3Es and/or at least 19% of the TE3s present in the microorganism as described herein, this inactivation is preferably associated with the absence of macroscopic disease symptoms.
By “macroscopic disease symptoms” is meant herein any disease symptom that can be seen with the naked eye (i.e., without magnification), such as a lesion, chlorosis, necrosis wilt, blight, spot, rot, galls, collapse, and the like. Macroscopic disease symptoms may more particularly be evaluated using a disease index.
A “disease index” notably indicates the degree of macroscopic symptoms associated with disease that can be observed in a patho-assay, in particular in an Arabidopsis plant, and which are ranked on a scale of 0 to X, with X corresponding to a number superior or equal to 4, preferably 4, 5, 9 or 10. In all cases, a score of 0 corresponds to the absence of macroscopic disease symptoms. When a disease index having a scale of 0 to 4 is used (such as that described in the Examples below), the scoring is as follows:
0: no macroscopic disease symptoms;
1 : chlorosis;
2: extended chlorosis;
3: necrosis;
4: leaf death.
Preferably, the scoring is as follows:
0: no macroscopic disease symptoms;
1 : chlorosis, in particular a yellow chlorosis at the inoculation point;
2: extended chlorosis, in particular wherein the yellow chlorosis reaches two inoculation points;
3: necrosis;
4: leaf death.
More preferably, the scoring is as follows:
0: no macroscopic disease symptoms;
0.5: green chlorosis at the inoculation point;
1 : yellow chlorosis at the inoculation point;
1.5: yellow chlorosis extended to the mesophyll;
2: yellow chlorosis reaching two inoculation points;
2.5: chlorosis covering more than 1 /3 of the leaf surface;
3: necrosis;
3.5: extended necrosis;
4: leaf death. The disease index is typically obtained by scoring multiple sites (e.g., multiple leaves), and obtaining the mean or median. More particularly, the score is obtained using the formula: (2 of scores of a given treatment group) / number of sites scored. Such a disease index is notably illustrated in Figure 4 of Meyer et al., 2005, incorporated herein by reference. The disease index may be scored directly or using photographs taken of the sites. When using photographs, the disease index may notably be scored at a resolution of 10 dpi. Preferably, said disease index is determined using the patho-assay as described in the Examples below (“Patho-assays” section). As a non-limiting example, the patho-assay may comprise i) performing wound inoculation of the main leaf vein of 4-week-old Arabidopsis plants with a bacterial suspension at 10 8 cfu/mL, and ii) evaluating the disease index of the inoculated leaf 24h to 48h after inoculation. Preferably, said disease index is scored on a scale of 0 to 4, more preferably as described herein.
When macroscopic disease symptoms are evaluated using a disease index scale, the Xanthomonas microorganism may have a score inferior to 2, equal or inferior to 1 , inferior to 1 , or equal to 0 on a disease index scale. The Xanthomonas microorganism may notably have no macroscopic disease symptoms.
Preferably, the Xanthomonas microorganism of the invention comprises the inactivation of at least five genes encoding T3Es secreted by the T3SS and has a score inferior to 2, preferably equal or inferior to 1 , more preferably inferior to 1 on a disease index scale. According to particularly preferred embodiment, the Xanthomonas microorganism has a score of 0 on a disease index scale. Said disease index preferably ranges from 0 to 4. Thus, the Xanthomonas microorganism preferably has no macroscopic disease symptoms (e.g., chlorosis, necrosis, lesions, etc.). Said disease index scale preferably ranges from 0 to 4, more preferably said disease index scale is that provided herein or that provided in Meyer et al., 2005.
The Xanthomonas microorganism in which at least five genes encoding type III effector proteins secreted by the type III secretion system are inactivated and comprising a functional type III secretion system as provided herein notably has the same level of pathogenicity as a corresponding Xanthomonas microorganism (i.e. , of the same species, and preferably of the same pathovar) in which the T3SS itself has been inactivated. Indeed, with each of these microorganisms, the disease index is less than 1 , preferably less than 0.5, even more preferably equal to 0. Thus, the Xanthomonas microorganism preferably has a disease index that is identical to that of a corresponding type III secretion mutant, more preferably a hrcV mutant.
“Type III secretion system” or “T3SS” refers herein to a set of structural proteins that are required to transport at least one polypeptide from the microorganism cytoplasm to a target plant cell. A functional T3SS is capable of moving at least at least one polypeptide to a target plant cell, and may notably comprise one or more chaperone proteins (e.g., HpaB). The skilled person is thus able to determine if a T3SS is functional based on its ability to transport at least one T3E. As an example, T3SS functionality can be shown by demonstrating type Ill-dependent in vitro secretion of T3E proteins (Rossier et al 1999) and translocation of T3E-reporter fusions which activity can be monitored in planta (Xu et al 2008). A hrcV is defective in type III secretion and may thus be used as a control to verify functionality (Hartmann and Buttner, 2013).
A functional T3SS may notably comprise at least the following nine Hrc (for “Hrp- conserved”) proteins: HrcC, HrcJ, HrcR, HrcS, HrpU, HrcT, HrcV, HrcQ and HrcN. A functional T3SS may also comprise other species-specific hrp genes which are by definition required for hypersensitive response (HR), pathogenicity, and T3E translocation (Bonas et al., 1991 ; Rossier et al., 1999). As a non-limiting example, the functional T3SS may further comprise at least one protein selected from among HrpB1 , HrpB2, HrpB4, HrpB5, HrpB7, HrpD5, HrpD6, HrpE1 , HrpF, and any combination of two or more thereof. The functional T3SS may notably comprise all of above mentioned Hrp proteins. A functional T3SS may further comprise the HpaM protein (Li et al., 2017). The minimal set of proteins required for a functional T3SS may, in some cases, vary according to the type of polypeptide being transferred, the Xanthomonas species, and/or the plant species. The functional T3SS may be encoded by a chromosomal hrp (hypersensitive response and pathogenicity) gene cluster (see e.g., Bonas et al., 1991 ). In particular, the proteins necessary for a functional T3SS may be coded within chromosomal hrp gene clusters, more particularly within at least the following six operons: hrpA, hrpB, hrpC, hrpD, hrpE, and hrpF.
Preferably, the functional T3SS comprises at least the HrcC, HrcJ, HrcR, HrcS, HrpU, HrcT, HrcV, HrcQ and HrcN proteins. Preferably, the functional T3SS further comprises other species-specific hrp genes which are by definition required for HR, pathogenicity and T3E translocation (Bonas et al., 1991 ; Rossier et al., 1999). Preferably, the functional T3SS further comprises at least one protein selected from among HrpB1 , HrpB2, HrpB4, HrpB5, HrpB7, HrpD5, HrpD6, HrpE1 , HrpF, and any combination of two or more thereof, more preferably a combination of all of said proteins. Preferably, the functional T3SS further comprises the HpaM protein. Preferably, the functional T3SS is encoded by chromosomal hrp gene clusters, more preferably by at least the hrpA, hrpB, hrpC, hrpD, hrpE, and hrpF operons.
Preferably, in the Xanthomonas microorganism provided herein, at least five genes encoding T3Es selected from the group consisting of: AvrBsI , AvrBs2, AvrBs3, XopB, XopC1 , XopC2, XopD, XopE1 , XopE2, XopE3, XopE4, XopE5, XopF1 , XopF2, XopF3, XopG1 , XopG2, XopG3, XopH1 , XopHZ, XopH2, XopH , Xopl2, XopJ1 , XopJ2, XopJ3, XopJ4, XopJ5, XopJ6, XopK, XopL, XopM, XopN, XopO, XopP, XopQ, XopR, XopS, XopT, Xopll, XopV, XopW, XopX, XopY, XopZ1 , XopZZ, XopAA, XopAB, XopAC, XopAD, XopAE, XopAFI , XopAFZ, XopAG, XopAH, XopAI, XopAJ, XopAK, XopALI , XopALZ, XopAM, XopAN, XopAO, XopAP, XopAQ, XopAR, XopAS, XopAT, XopAU, XopAV, XopAW, XopAX, XopAY, XopAZ, XopBA, XopA, HpaA, HrpW, AvrXccAI , AvrXccAZ, any variant thereof, and any fragment thereof, are inactivated.
Preferably, said fragment or variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identify with the corresponding type III effector protein. More preferably, in the Xanthomonas microorganism provided herein, the at least five genes encode T3Es selected from the group consisting of: AvrBsI , AvrBsZ, AvrBs3, XopB, XopC1 , XopCZ, XopD, XopE1 , XopEZ, XopE3, XopE4, XopE5, XopF1 , XopFZ, XopF3, XopG1 , XopGZ, XopG3, XopH1 , XopHZ, XopHZ, Xopll , XopIZ, XopJ1 , XopJZ, XopJ3, XopJ4, XopJ5, XopJ6, XopK, XopL, XopM, XopN, XopO, XopP, XopQ, XopR, XopS, XopT, XopU, XopV, XopW, XopX, XopY, XopZ1 , XopZZ, XopAA, XopAB, XopAC, XopAD, XopAE, XopAFI , XopAFZ, XopAG, XopAH, XopAI, XopAJ, XopAK, XopAH , XopALZ, XopAM, XopAN, XopAO, XopAP, XopAQ, XopAR, XopAS, XopAT, XopAU, XopAV, XopAW, XopAX, XopAY, XopAZ, XopBA, XopA, HpaA, HrpW, AvrXccAI , and AvrXccAZ.
Preferably, in the Xanthomonas microorganism provided herein, at least five genes encoding T3Es selected from the group consisting of: AvrBsI , AvrXccAI , AvrXccAZ, HrpW, XopAC, XopAG, XopAH, XopALI , XopALZ, XopAM, XopAN, XopD, XopEZ, XopF, XopG, XopH, XopJ5, XopK, XopL, XopN, XopP, XopQ, XopR, XopX1 , XopXZ, and XopZ are inactivated. Preferably, the at least five genes encoding T3Es that are inactivated in the Xanthomonas microorganism comprise xopAC, avrBsI , xopH, xopX1 , and xopXZ.
Preferably, at least 11 genes encoding T3Es are inactivated in the Xanthomonas microorganism, wherein the at least 11 genes comprise xopAC, avrBsI , xopH, xopX1 , xopXZ, xopF, hrpW, xopD, xopAN, xopQ, and xopK. Preferably, at least 1Z genes encoding T3Es are inactivated in the Xanthomonas microorganism, wherein the at least 1Z genes comprise xopAC, avrBsI , xopH, xopX1 , xopXZ, xopF, hrpW, xopD, xopAN, xopQ, xopK, and xopJ5. Preferably, at least ZZ genes encoding T3Es are inactivated in the Xanthomonas microorganism, wherein the at least ZZ genes comprise xopAC, avrBsI , xopH, xopX1 , xopXZ, xopF, hrpW, xopD, xopAN, xopQ, xopK, xopJ5, xopG, avrXccAI , avrXccAZ, xopAM, xopEZ, xopAH, xopR, xopL, xopZ, and xopAG. Preferably, at least Z5 genes encoding T3Es are inactivated in the Xanthomonas microorganism, wherein the at least Z5 genes comprise xopAC, avrBsI , xopH, xopX1 , xopXZ, xopF, hrpW, xopD, xopAN, xopQ, xopK, xopJ5, xopG, avrXccAI , avrXccAZ, xopAM, xopEZ, xopAH, xopR, xopL, xopZ, xopAG, xopP, xopALI , and xopALZ. According to a particularly preferred embodiment, in the Xanthomonas microorganism provided herein, the genes encoding the following T3Es: AvrBsI , AvrXccAI , AvrXccAZ, HrpW, XopAC, XopAG, XopAH, XopALI , XopAL2, XopAM, XopAN, XopD, XopE2, XopF, XopG, XopH, XopJ5, XopK, XopL, XopN, XopP, XopQ, XopR, XopX1 , XopX2, and XopZ, are inactivated. Preferably, the above-listed T3Es have the amino acid sequences as provided in SEQ ID NOs: 29 to 54, respectively.
As a non-limiting example, the at least five genes encoding T3Es which are inactivated are selected from the genes having the sequences as provided in SEQ ID NOs: 1 to 26, any variant thereof, and any fragment thereof. The above definitions and preferred embodiments related to the fragments and variants of proteins apply mutatis mutandis to nucleotide sequences, such as genes, encoding said proteins. Preferably, the at least five genes encoding T3Es which are inactivated are selected from the genes having the sequences as provided in SEQ ID NOs: 1 to 26, any variant thereof, and any fragment thereof. More preferably, the at least five genes encoding T3Es which are inactivated are selected from the genes having the sequences as provided in SEQ ID NOs: 1 to 26. Even more preferably, the genes encoding T3Es having the sequences as provided in SEQ ID NOs: 1 to 26 are inactivated.
The Xanthomonas microorganism may notably correspond to the microorganism having the identification reference number “Xcc 8004 DELTA 26T3E” which was deposited at the “Collection Nationale de Cultures de Microorganismes” (CNCM) of the Institut Pasteur, 25-28 rue du Docteur Roux, 75724, Paris, France (an International Depository Authority under the Budapest Treaty), on July 1 , 2020, having the registration number I- 5530.
Thus, according to a preferred embodiment, the Xanthomonas microorganism is the microorganism deposited at the CNCM under registration number I-5530 on July 1 , 2020.
According to a further aspect, the present invention relates to a Xanthomonas microorganism as described according to any of the above embodiments, which further comprises at least one gene coding for a heterologous type III effector protein. Indeed, the effect of a heterologous T3E on Xanthomonas pathogenicity, host specificity, etc. may be advantageously be evaluated in a microorganism comprising a heterologous gene encoding said T3E, with no restrictions as to the host plant and/or T3E that is selected.
A “heterologous” gene as used herein refers to a gene encoding a protein or polypeptide that is introduced into a microorganism in which said gene does not naturally occur. A “heterologous” gene as used herein also encompasses a gene that was endogenous (or native) to a microorganism (i.e., present in the microorganism prior to any genetic modification) but that, when introduced into the microorganism, is not introduced at the location where the endogenous gene was located.
A heterologous gene may be directly integrated into the chromosome of the microorganism, e.g., by genetic recombination, or be expressed extra-chromosomally within the microorganism as part of a plasmid or vector. For successful expression, the heterologous or exogenous gene(s) may be introduced into the microorganism with all of the regulatory elements necessary for their expression or introduced into a microorganism that already comprises all of the regulatory elements necessary for their expression.
As a non-limiting example, the Xanthomonas microorganism may further comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, or more heterologous genes encoding T3Es. Said one or more heterologous genes encoding T3Es may be selected from the group consisting of AvrBsI , AvrBs2, AvrBs3, XopB, XopC1 , XopC2, XopD, XopE1 , XopE2, XopE3, XopE4, XopE5, XopF1 , XopF2, XopF3, XopG1 , XopG2, XopG3, XopH1 , XopH2, XopH2, Xopll , Xopl2, XopJ1 , XopJ2, XopJ3, XopJ4, XopJ5, XopJ6, XopK, XopL, XopM, XopN, XopO, XopP, XopQ, XopR, XopS, XopT, Xopll, XopV, XopW, XopX, XopY, XopZ1 , XopZ2, XopAA, XopAB, XopAC, XopAD, XopAE, XopAFI , XopAFZ, XopAG, XopAH, XopAI, XopAJ, XopAK, XopALI , XopALZ, XopAM, XopAN, XopAO, XopAP, XopAQ, XopAR, XopAS, XopAT, XopAU, XopAV, XopAW, XopAX, XopAY, XopAZ, XopBA, XopA, HpaA, HrpW, AvrXccAI , AvrXccAZ, variants thereof, and fragments thereof.
According to a preferred embodiment, the Xanthomonas microorganism may further comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, or more heterologous genes encoding T3Es. Preferably, the Xanthomonas microorganism further comprises a heterologous gene encoding a T3E selected from the group consisting of AvrBsI , AvrBs2, AvrBs3, XopB, XopC1 , XopC2, XopD, XopE1 , XopE2, XopE3, XopE4, XopE5, XopF1 , XopF2, XopF3, XopG1 , XopGZ, XopG3, XopH1 , XopH2, XopH2, Xopll , Xopl2, XopJ1 , XopJ2, XopJ3, XopJ4, XopJ5, XopJ6, XopK, XopL, XopM, XopN, XopO, XopP, XopQ, XopR, XopS, XopT, XopU, XopV, XopW, XopX, XopY, XopZ1 , XopZ2, XopAA, XopAB, XopAC, XopAD, XopAE, XopAFI , XopAFZ, XopAG, XopAH, XopAI, XopAJ, XopAK, XopAH , XopAL2, XopAM, XopAN, XopAO, XopAP, XopAQ, XopAR, XopAS, XopAT, XopAU, XopAV, XopAW, XopAX, XopAY, XopAZ, XopBA, XopA, HpaA, HrpW, AvrXccAI , AvrXccAZ, variants thereof, fragments thereof, and any combination of two or more thereof. More preferably, the Xanthomonas microorganism further comprises 1 , 2, 3, 4, or 5 heterologous genes encoding T3Es, even more preferably selected from those described herein. Preferably, the number of heterologous genes present in the Xanthomonas microorganism remains inferior to the number of genes encoding T3Es which were inactivated in said microorganism. As a nonlimiting example, if the Xanthomonas microorganism comprises the inactivation of five genes encoding T3Es, it further comprises 1 , 2, 3, or 4 heterologous genes encoding T3Es.
In cases where the selected heterologous gene encoding a T3E was previously deleted in the Xanthomonas microorganism, the reintroduction of said gene is such that it is not located at the same location as the endogenous gene which was initially present.
Preferably, the heterologous gene is a gene that does not naturally occur in the microorganism. Preferably, the heterologous gene is a gene that naturally occurs in the microorganism but that has been reintroduced at a position different to that of the naturally occurring gene, more preferably, said endogenous gene has been inactivated.
According to a further aspect, the present invention relates to a use of the Xanthomonas microorganism as provided herein to evaluate the recognition of at least one type III effector protein by a plant, wherein the microorganism comprises at least one gene encoding a heterologous type III effector protein or a variant or fragment thereof.
The term “plant” as used herein refers to any representative of the Plantae kingdom. Said plant may be an angiosperm. Said plant may be a monocot or a dicot. Said plant may be known to be susceptible to Xanthomonas or not. The plant may be genetically modified (i.e. , comprising a genetic construct such as a "transgene" that is not found in wild-type plants of the same species or variety) or not. Said plant may more particularly be selected from among the monocot families Amatyllidaceae, Araceae, Arecaceae, Asparagaceae, Cannaceae, Iridaceae, Liliaceae, Musaceae, and Poaceae, or from among the dicots families Anacardiaceae, Apiaceae, Araliceae, Asteraceae, Atherospermataceae, Begoniaceae, Betulaceae, Brassicaceae, Cannabaceae, Cucurbitaceae, Ebenaceae, Euphorbiaceae, Fabaceae, Geraniaceae, Juglandaceae, Lamiaceae, Lythraceae, Malvaceae, Martyniaceae, Meliaceae, Monimiaceae, Myrtaceae, Oleaceae, Oxalidaceae, Papaveraceae, Pedaliaceae, Phyllanthaceae, Piperaceae, Plantaginaceae, Rosaceae, Rubiaceae, Rutaceae, Salicaceae, Solanaceae, Theaceae, Verbenaceae, and Vitaceae. Preferably, said plant is a whole plant, though plant parts, such as leaves, stems, roots (including tubers), flowers, tissues, and fruit, may also be envisaged. Said plant includes ancestors and progeny of a given plant or plant part. The plant may be grown to produce edible roots, tubers, leaves, stems, flowers, or fruits.
The plant may more particularly be a crop plant (e.g., cereals and legumes, maize, wheat, potato, cassava, rice, sorghum, millet, cassava, barley, or peas) or other vegetables or fruit plants, such as stone fruits. As a non-limiting example, the crop plant may be corn (maize (Zea mays)), rape (Brassica napus, Brassica rapa), mustard (Brassica juncea), flax (Flax usittissimum), alfalfa (Alfalfa sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus), wheat (Tritium aestivum), soybean (Glycine max), tobacco (Nicotiana spp. ), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea batatas), cassava (Manihot esculenta), tea (Camellia sinensis), coffee (Coffea), oats (Avena sativa), or barley (Hordeum vulgare). The plant may be a citrus, such as grapefruit (Citrus x paradisi), Key lime (C. aurantii folia), Kaffir lime (C. hystrix), lemon (C. limon)), trifoliate orange (Poncirus trifoliata), or bitter orange (C. aurantium). The plant may be a nut or fruit, such as avocado (Persea americana), fig (Ficus carica), guava (Psidium guajava), mango (Mangifera spp), papaya (Carica papaya), banana ( usa), cashew (Anacardium occidentale), walnut (Juglans), almond (Prunus dulcis), wine grape (Vitis vinifera), peach (Prunus persica), plum (Prunus spp.), or pear (Pyrus). The plant may be a vegetable, such as a pepper (Capsicum spp.), tomato (Solanum lycopersicum), common bean (Phaseolus vulgaris), beet (Beta vulgaris), lettuce (Lactuca sativa), chicory (Cichorium intybus), cruciferous vegetable of the Brassica genus (such as cabbage, broccoli, turnip, rutabaga, or cauliflower), gourd (Lagenaria spp. or Cucurbita spp.), carrot (Daucus carota), strawberry (Fragaria x ananassa), and the like. In particular, said plant may be a pepper, tomato, brassica, rice, wheat, barley, citrus, banana, cassava, peanut, cotton, lettuce, walnut, strawberry, or bean plant.
Preferably, said plant is selected from any of those provided in the previous paragraph. More preferably, said plant is a pepper, tomato, brassica, rice, wheat, barley, citrus, banana, cassava, peanut, cotton, lettuce, walnut, strawberry, or bean plant.
“Recognition” of a T3E by a plant means herein that the plant detects and reacts to said T3E. Said recognition may induce a change in transcription, translation, DNA fragmentation, metabolite production (e.g., production of reactive oxygen species (ROS)), etc, which may or may not be associated with a macroscopic change which is visible to the naked eye. Preferably, recognition causes macroscopic change. As a non-limiting example, the macroscopic change may be the presence of lesions, chlorosis, wilt, blight, spot, rot, galls, necrosis, collapse, etc. In particular, recognition of a T3E by a plant may be determined by macroscopic collapse, necrosis or cell death of the infected tissues. The most extreme expression of the recognition of a plant pathogen is the hypersensitive response (HR) or HR-like reactions which, in addition to plant cell death also restrict(s) pathogen multiplication and systemic spread. The HR may notably include ion leakage, tissue collapse, ROS production, etc. Recognition of a T3E by a plant may be mediated by corresponding resistance (R) genes or R proteins of the plant and induce effector-triggered immunity (ETI). Recognition may be evaluated using a patho-assay known to the skilled person or according to the methods described herein. As a non-limiting example, recognition of a T3E by a plant may be evaluated by inoculating a plant with a suspension of Xanthomonas microorganism comprising at least one heterologous T3E and determining if one or more macroscopic changes result after a given amount of time has passed (e.g., 24 or 48 hours, 1 , 2, or 3 weeks). Macroscopic changes (or symptoms) may notably can be compared to one or more control plants which have not been inoculated with a Xanthomonas microorganism comprising at least one heterologous T3E, or which have been inoculated with a Xanthomonas microorganism that does not cause macroscopic disease symptoms or which has a score of 0 on a disease index, or photo(s) thereof (i.e. negative control) or control plants which have been inoculated with a wild-type Xanthomonas microorganism (i.e. positive control).
As a non-limiting example, recognition of a T3E by a cauliflower plant may be evaluated by performing infiltration of the leaf mesophyll of 4-week-old cauliflower plants with a bacterial suspension at 10 8 cfu/mL, and evaluating the disease index of the inoculated leaf area 24h to 48h after inoculation (e.g., using a disease index having a scale of 0 to 4, as described herein).
According to a further aspect, the present invention further relates to a use of the Xanthomonas microorganism expressing at least one heterologous gene encoding a type III effector protein or a variant or fragment thereof as provided herein to evaluate the tolerance or resistance of a plant to the Xanthomonas microorganism when the type III effector protein is recognized by the plant.
“Tolerance” of a plant as used herein means that plant host fitness costs caused by Xanthomonas are reduced or alleviated when compared to susceptible plant varieties under similar environmental conditions and Xanthomonas pressure (e.g., similar bacterial load). Preferably no macroscopic disease symptoms are detected in the plant. Nevertheless, multiplication of Xanthomonas microorganism is detected in infected tissues. Xanthomonas multiplication may be evaluated using a bacterial in planta growth assay. Such assays are well-known in the art. As a non-limiting example, when the tissue is a leaf, leaf punches may be collected, homogenized, diluted, and plated on a solid medium (e.g., NYG agar), and the number of colonies counted 2-3 days after plating. Leaf punches, or other plant tissue samples, may be collected at one or more time points ranging from 24 hours to 14 days after the plant has been inoculated. Bacterial growth levels may be compared between samples collected from plants which have been inoculated with a Xanthomonas microorganism comprising at least one heterologous T3E as provided herein and one or more control plants, such as those described above. Control plants are preferably inoculated with a wild-type Xanthomonas microorganism (thus providing a positive control).
“Resistance” of a plant as used herein means that plant host fitness costs caused by Xanthomonas are reduced or alleviated by inhibiting Xanthomonas infection or that a plant lacks susceptibility to Xanthomonas. Resistant plants may not display macroscopic disease symptoms. Xanthomonas microorganism multiplication and systemic spread is reduced compared to susceptible cultivars of the host plants under similar environmental conditions and Xanthomonas pressure. Resistant varieties may nevertheless exhibit some disease symptoms or damage under heavy Xanthomonas pressure. Two levels of resistance may notably be observed: high resistance and intermediate resistance. High resistance occurs when a plant variety highly restricts the growth and/or development of Xanthomonas and/or the damage it causes under normal pressure (e.g., bacterial load) when compared to susceptible varieties. Highly resistant plant varieties may, however, exhibit some symptoms or damage under heavy Xanthomonas pressure. Intermediate resistance occurs when a plant variety restricts the growth and/or development of the specified pest and/or the damage it causes but may exhibit a greater range of symptoms or damage when compared to high resistant varieties. Nevertheless, plants with intermediate resistance will still show less severe symptoms or damage than susceptible varieties when grown under similar environmental conditions and/or Xanthomonas pressure. Preferably, resistant plants do not display macroscopic disease symptoms. Preferably, resistant plants show very reduced bacterial multiplication (e.g., reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% as compared to a susceptible cultivar of the host plant) in a bacterial growth assay, such as that provided herein. More preferably resistant plants show no bacterial multiplication in a bacterial growth assay, such as that provided herein.
Tolerance and/or resistance of a plant to Xanthomonas infection may be evaluated by measuring the level of effector-triggered immunity, which is accompanied by localized cell death, associated tissue collapse known as the hypersensitive response (HR) at the site of infection, and limited spread of the pathogen. As a non-limiting example, tolerance and/or resistance of a plant to Xanthomonas infection may be determined by measuring gene expression levels, electrolyte leakage, cell death, DNA fragmentation, production of ROS in a plant, or by determining its Ca 2+ signature. Such techniques are well-known to the person skilled in the art.
According to a further aspect, the present invention relates to a method for the evaluation of the recognition of a type III effector protein by a plant comprising the steps of: a) introducing a heterologous gene coding for the type III effector protein into the Xanthomonas microorganism as provided herein, b) inoculating a plant with an appropriate amount of the Xanthomonas microorganism obtained in step a), and c) evaluating the recognition of the type III effector protein by the plant.
The term “inoculating” as used herein refers to bringing Xanthomonas in contact with plant tissue. Methods of inoculating Xanthomonas are well-known in the art, and include, as a non-limiting example, directly dropping a suspension of Xanthomonas on a plant tissue, injecting a Xanthomonas bacterial suspension into the tissue (e.g. wounding of a plant with a needle dipped in a bacterial suspension), immersing the plant tissue in the Xanthomonas suspension, pressurized spraying, multiple pricking, carborundum abrasion, clipping a leaf and exposing crosscut veins to a Xanthomonas suspension, introducing a Xanthomonas suspension into guttation droplets, etc.
Reference to an “appropriate amount” as used herein refers to the amount of Xanthomonas microorganism capable of inducing a response (e.g., a hypersensitive reaction) in a plant when the plant is not tolerant or resistant to Xanthomonas. As a nonlimiting example, a bacterial suspension of 10 5 to 10 9 CFU/mL may be used to inoculate a plant site, more particularly a suspension of 10 8 to 4 x10 8 CFU/mL may be used to inoculate a plant site.
Recognition may be evaluated using a patho-assay known to the skilled person or according to the methods described herein.
Preferably, the method further comprises a step of: d) evaluating the tolerance or resistance of the plant to the Xanthomonas microorganism when the type III effector protein is recognized by the plant.
Tolerance or resistance may be evaluated using a method known to the skilled person or according to a method described herein.
The plant may be any plant provided herein. Preferably, the plant is a monocotyledonous or dicotyledonous plant, preferably a pepper, tomato, brassica, rice, wheat, barley, citrus, banana, cassava, peanut, cotton, lettuce, walnut, strawberry, or bean plant.
Figures
Figure 1 : The polymutant 8004A26T3E of Xanthomonas campestris pv. campestris does not cause disease symptoms on Arabidopsis thaliana accession Sf2 similar to the type III secretion mutant 8004AhrcV. Four-week-old plants were inoculated by piercing the central vein with a needle dipped in a bacterial suspension at 10 8 cfu/mL. Disease index was scored at 10 days post-inoculation (dpi) using the following scoring: 0: no symptoms; 1 : chlorosis; 2: extended chlorosis; 3: necrosis; 4: leaf death. Three independent experiments were performed and were combined. Boxplot representations are as follows: middle bar: median; box limits: upper and lower quartiles; extremes: minimum and maximum values. Dots indicate outliers.
Figure 2: The polymutant 8004A26T3E from Xanthomonas campestris pv. campestris is able to translocate XopJ6cN06, an effector from Xcc strain CN06 recognized in cauliflower cv. Clovis F1. Leaves of Brassica oleracea var. botrytis cv Clovis F1 were infiltrated with bacterial suspensions at 10 8 cfu/mL and collapse was observed after 24 hours. This rapid tissue collapse is a hallmark of extreme ETI responses referred to as hypersensitive response. Strain 8004 and its polymutant 8004A26T3E were not able to induce a collapse. The natural CN06 strain and the 8004A26T3E xopJ6 induce a collapse visible by the lighter grey shading in those infiltrated areas of the leaves (delimited by dashed white lines).
Figure 3: The polymutant 8004A26T3E from Xanthomonas campestris pv. campestris is able to translocate AvrBsI soo4, an effector from Xcc strain 8004 recognized by Bs1 resistance gene in nonhost pepper plant ECW-10R. Leaves of Capsicum annuum ECW-10R were infiltrated with bacterial suspensions at 2x10 8 cfu/mL and collapse was observed after 48 hours. This rapid tissue collapse is a hallmark of extreme ETI responses referred to as hypersensitive response (HR). The HR triggered by wild-type (WT) strain 8004 depends on avrBsI and a functional T3SS (8004AhrcV mutant). Polymutant 8004A26T3E carrying the plasmid pDSK-EV (empty vector) is deleted for avrBsI and does not induce HR. Strain 8004A26T3E pDSK-avrBs/sotM triggers HR. Areas of tissue collapse are delimited by dashed white lines.
Examples
The present invention is further defined in the following examples. It should be understood that these examples, while indicating preferred embodiments of the invention, are given by way of illustration only. The person skilled in the art will readily understand that these examples are not limitative and that various modifications, substitutions, omissions, and changes may be made without departing from the scope of the invention.
Methods
Strains and growth conditions Xanthomonas campestris pv. campestris (Xcc) strains 8004 (Daniels et al., 1984) and CFBP6943 (http://catalogue-cfbp.inra. fr/resultnum_e.php?rO=6943) and Xanthomonas euvesicatoria (Xe, previously known as Xanthomonas campestris pv. vesicatoria) strain 85- 10 (Bonas et al., 1989) were grown at 28 °C in MOKA medium (Blanvillain et al., 2007). Escherichia coli cells were grown on Luria-Bertani medium at 37° C. For solid media, agar was added at a final concentration of 1.5% (wt/vol). Antibiotics were used at the following concentrations: for Xcc, 50 pg/mL rifampin, 50 pg/mL kanamycin and 40 pg/mL spectinomycin. For E. coli, 50 pg/mL kanamycin and 40 pg/mL spectinomycin.
Plant material and growth conditions
The following plant accessions were used: Brassica oleracea cv. Clovis F1 (Vilmorin SA) is a host plant for Xcc (Cerutti et al., 2017) and responds by a hypersensitive response (HR) upon XopJ6 recognition. Arabidopsis thaliana accession Sf-2 is a host plant to Xcc and is susceptible to infection by Xcc strain 8004. Peppers (Capsicum annuum) are not a host plant for Xcc strains (Vicente et al., 2013). Accessions ECW-10R and ECW-30R are near isogenic lines expressing Bs1 and Bs3 resistance genes, respectively, resulting in HR induction upon detection of Xe AvrBsI and Xe AvrBs3 T3E proteins (Hibberd et al., 1987; Bonas et al., 1989).
Arabidopsis plants were grown in Jiffy pots in a growth chamber at 22 °C, with a 9-h light period and a light intensity of 192 pmol m' 2 s' 1 . Brassica and pepper plants were grown under greenhouse conditions.
Sequential deletion of T3E genes in Xanthomonas campestris pv. campestris
Deletion mutants in Xcc strain 8004 were obtained using the SacB system with a pA13 suicide vector (Guy et al., 2013). pA13 is a derivative of the pK18 suicide vector (Guy et al., 2013) modified for GoldenGate cloning (Engler et al., 2008). Deletion of the entire coding sequences of the following genes was achieved: XC_0241 , XC_0268, XC_0541 , XC_0542, XC_0563, XC_0967, XC_1210, XC_1213, XC_1553, XC_1716, XC_2004, XC_2081 , XC_2082, XC_2210, XC_2602, XC_2994, XC_2995, XC_3024, XC_3023, XC_3160, XC_3176, XC_3177, XC_3802, XC_3915-6, XC_4273, and XC_4318, corresponding to SEQ ID NOs 1 -26, respectively. All pA13 plasmid derivatives used for deletions were previously described (Guy et al. , 2013) except for xopAG, xopAN and xopAL2 which were designed in a similar manner. Plasmids were introduced into E. coli by electroporation and into Xcc by triparental mating as previously described (Figurski and Helinski, 1979; Ditta et al., 1980). Deletion events were selected as described (Schafer et al., 1994) and verified by PCR. Deletion of the 26 genes mentioned above was confirmed in the final 8004A26T3E strain by whole genome sequencing using single molecule-long read technology.
Re-introduction of Xanthomonas T3E genes in Xcc 8004A26T3E xopJ6xccCN06 promoter and coding regions were cloned into the pCZ1301 integrative vector, which is a derivative of the pK18 suicide plasmid (Schafer et al., 1994) in which the multicloning site is flanked by 500-bp-long genomic regions of Xcc strain 8004 allowing targeted and stable insertion of sequences into the genome of strain 8004 by double homologous recombination. pENTRY vector containing avrBs3xe75-3 was recombined into pDSK-GW destination vectors by LR recombination yielding the pDSK-avrBs3xe75-3 expression vector.
All plasmids were introduced into Xcc strain 8004A26T3E by triparental mating as described (Figurski and Helinski, 1979; Ditta et al., 1980). Genomic integration was selected as described (Schafer et al., 1994). All final strains were verified by PCR.
Patho-assays
Xcc inoculations were performed on 4-week-old plants in growth chambers (9 h light; 22 °C; relative humidity 70%). Bacteria were harvested from overnight cultures in MOKA by centrifugation (4,000g, 10 min) and suspended in 1 mM MgCL.
On Arabidopsis, Xcc pathogenicity was assayed by wound inoculation of the main leaf vein of 4-week-old Arabidopsis plants with a bacterial suspension at 10 8 cfu/mL essentially as described in Meyer et al., 2005. The disease index was scored at 10 dpi on a 0 to 4 scale as follows: 0: no macroscopic disease symptoms; 1 : chlorosis; 2: extended chlorosis; 3: necrosis; 4: leaf death.
Complementary tests allowed for a more precise determination of the disease index, using the following scale: 0: no macroscopic disease symptoms; 0.5: green chlorosis at the inoculation point; 1 : chlorosis (more particularly yellow chlorosis at the inoculation point); 1.5: yellow chlorosis extended to the mesophyll; 2: extended chlorosis (more particularly yellow chlorosis reaching two inoculation points); 2.5: chlorosis covering more than 1 /3 of the leaf surface; 3: necrosis; 3.5: extended necrosis; 4: leaf death.
Effector translocation assays were performed by following tissue collapse after infiltration of mesophyll tissue of pepper or cauliflower leaves using a needleless syringe as described in Xu et al., 2008. The infiltrated area was marked with a black marker on the leaf. Pictures were taken 24h to 48h after infiltration.
Results
Construction of strain 8004A26T3E by serial deletion of T3E genes T3E genes were deleted sequentially from a wild-type Xcc strain 8004 in 21 steps due the presence of five effector gene pairs. Deletions were confirmed in 8004A26T3E by PCR using primers flanking the deletions and complete genome sequencing using long-read technology (data not shown). The integrity of the hrp gene cluster was confirmed by the absence of undesired mutations.
Evaluation of the pathogenicity of intermediate deletion mutants
Pathogenicity of intermediate deletion mutants of Xcc strain 8004 comprising the deletion of various intermediate numbers of genes coding for T3Es was evaluated on Brassica oleracea cv. Clovis F1 or Arabidopsis thaliana accession Sf-2 10 days after inoculation using a disease index having a scale of 0 to 4. Results are presented in the table below, and correspond to the median of at least three independent biological replicates. The intermediate delete mutants are as follows:
Xcc strain 8004A5T3E: xopAC, avrBsI , xopH, xopX1 , and xopX2 deletions
Xcc strain 8004A11T3E: xopAC, avrBsI , xopH, xopX1 , xopX2, xopF, hrpW, xopD, xopAN, xopQ, and xopK deletions
Xcc strain 8004A12T3E: xopAC, avrBsI , xopH, xopX1 , xopX2, xopF, hrpW, xopD, xopAN, xopQ, xopK, and xopJ5 deletions
Xcc strain 8004A22T3E: xopAC, avrBsI , xopH, xopX1 , xopX2, xopF, hrpW, xopD, xopAN, xopQ, xopK, xopJ5, xopG, avrXccAI , avrXccAZ, xopAM, xopE2, xopAH, xopR, xopL, xopZ, and xopAG deletions
Xcc strain 8004A25T3E: xopAC, avrBsI , xopH, xopX1 , xopX2, xopF, hrpW, xopD, xopAN, xopQ, xopK, xopJ5, xopG, avrXccAI , avrXccAZ, xopAM, xopE2, xopAH, xopR, xopL, xopZ, xopAG, xopP, xopAH , and xopALZ deletions
Table 1 : Pathogenicity of intermediate deletion mutants As illustrated in the table above, an Xcc intermediate deletion mutant in which 5 genes coding T3Es were deleted had a significant reduction in pathogenicity in Brassica oleracea. Indeed, the disease index was reduced from approximately 3 to 2.5. An Xcc intermediate deletion mutant in which 11 genes coding T3Es were deleted showed a further reduction in pathogenicity, as the disease index was further reduced to approximately 1.5. A similar level of reduced pathogenicity was observed on Arabidopsis with an Xcc intermediate deletion mutant in which 12 genes coding T3Es were deleted (disease index of approximately 1.5). Xcc intermediate deletion mutants in which 22 genes coding T3Es were deleted had a disease index of 1 on Brassica and on Arabidopsis).
Xcc strain 8004A26T3E is non-pathogenic on Arabidopsis
Pathogenicity of Xcc strain 8004A26T3E was tested on susceptible host Arabidopsis thaliana accession Sf-2. 10 days post inoculation in the central vein by wounding, the WT strain is able to cause strong necrosis (Figure 1 ). The 8004A26T3E strain is unable to cause any disease symptoms similar to the 8004AhrcV mutant which is unable to secrete and translocate any T3E proteins. These results indicate that the 26T3E proteins are collectively essential for pathogenicity on Arabidopsis thaliana and mimic phenotypically the loss of the type 3 secretion machinery.
Strain Xcc 8004A26T3E can deliver Xcc effector XopJ6 in the host cauliflower plant Clovis F1
In order to test for the functionality of the type 3 secretion system in strain 8004A26T3E, we stably inserted in its genome the entire xopJ6 gene (promoter included) from Xcc strain CN06. As shown in Figure 2, strain CN06, when introduced in the mesophyll of Brassica oleracea var. botrytis cv Clovis F1 , causes a rapid tissue collapse caused by XopJ6 translocation and recognition inside plant cells. Strain 8004A26T3E: :xopJ6cN06 causes a strong tissue collapse characteristic of a hypersensitive response (HR) in contrast to strains 8004 or 8004A26T3E which do not express xopJ6cN06. These results demonstrate that 8004A26T3E has retained its ability to deliver a Xcc type 3 effector inside the plant cells of a host plant.
Strain Xcc 8004A26T3E can deliver Xcc effector AvrBsI in the nonhost pepper plant ECW-10R
In order to test if strain 8004A26T3E can deliver T3Es in nonhost plants, we overexpressed the avrBs oM gene from Xcc strain 8004 in strain 8004A26T3E. AvrBsI T3E from various Xanthomonas is recognized by the pepper Bs1 R gene present in pepper cultivar ECW-10R (Hibberd et al. 1987; Bonas et al., 1989, Xu et al., 2018). As shown in Figure 3, strain 8004, when introduced in the mesophyll of ECW-10R leaves, causes a rapid HR. Because strains lacking avrBsI (8004AavrBs1) or a functional T3SS (8004AhrcV) failed to induce HR, we conclude that HR is caused by T3SS-dependent AvrBsI translocation and recognition inside plant cells by Bs1 as previously reported. AvrBs1soo4 expression in strain 8004A26T3E (strain 8004A26T3E pDSK-avrBslaocw) triggered HR in contrast to the empty vector (EV) control (strain 8004A26T3E pDSK-EV). This indicates that strain 8004A26T3E retained the ability to translocate the AvrBsI T3E protein and that T3E translocation was possible into a nonhost plant.
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