Fungal rust-inducible promoter

Abstract

The disclosure relates to a promoter induced by fungal rust. More specifically, the promoter is induced by the pathogen Phakopsora pachyrhizi, i.e. the Asian Soybean Rust. The disclosure further provides for recombinant genes comprising the promoter and methods of producing a transgenic plant that involves introducing or providing the recombinant gene to plant cells to create transgenic plant cells, and regenerating transgenic plants from the transgenic cells.

Claims

1. A recombinant expression cassette comprising the nucleotide sequence of SEQ ID NO: 1 or a functional fragment thereof, operably linked to a heterologous nucleic acid encoding an expression product of interest, and optionally a transcription termination and polyadenylation sequence functional in plants.

2. The recombinant expression cassette according to claim 1, wherein the expression product of interest is a protein, or an RNA molecule capable of modulating the expression of a gene.

3. A soybean host cell, comprising the recombinant expression cassette according to claim 1.

4. A soybean plant, comprising the recombinant expression cassette of claim 1.

5. Plant parts and seeds obtainable from the plant according to claim 4.

6. A method of producing a transgenic soybean plant, comprising the steps of: a) introducing or providing the recombinant expression cassette according to claim 1 to a plant cell to create transgenic plant cells; and b) regenerating transgenic plants from said transgenic cells.

7. A method of effecting fungal rust-inducible expression of a nucleic acid, comprising introducing the recombinant expression cassette according to claim 1 into the genome of a plant.

8. The recombinant expression cassette according to claim 1, wherein the recombinant expression cassette comprises the nucleotide sequence of SEQ ID NO: 1.

9. The recombinant expression cassette according to claim 1, wherein the recombinant expression cassette comprises a functional fragment of SEQ ID NO: 1.

10. A method of conferring, in a soybean cell, expression of an expression product of interest in response to exposure of the soybean cell to a fungal rust, comprising providing, in the soybean cell, a nucleic acid, wherein the nucleic acid comprises a promoter comprising the nucleotide sequence of SEQ ID NO: 1 or a functional fragment thereof, operably linked to a nucleic acid sequence encoding the expression product of interest, wherein the nucleic acid sequence encoding the expression product of interest is heterologous to the promoter.

11. A soybean plant exhibiting fungal rust inducible expression of an expression product of interest, wherein the soybean plant comprises, in the genome of at least a cell, the recombinant expression cassette according to claim 1.

12. The soybean plant of claim 11, wherein the expression product of interest is a protein, or an RNA molecule capable of modulating the expression of a gene.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Accumulation of chitinase gene transcript in soybean leaf during P. pachyrhizi infection. Transcript abundance (via qRT-PCR) from individual leaves of soybean after P. pachyrhizi inoculation at 0, 8, 24, 48 and 72 hour post infection (hpi). The chitinase gene level is compared to reference mRNA levels and then normalized by mock treatment at each measurement time. Mock and P. pachyrhizi inoculations are realized on the same leaf. Bars indicate standard error of 4 biological replicates, Stars represent a significant difference compared to 0 hpi, using Students t-test (*for p<0.05; **for p<0.01).

(2) FIG. 2: Induction of chitinase gene promoter during P. pachyrhizi infection. Relative fluorescence intensity quantification of WT and Promoter-GFP (event 133 and 129) leaves on pathogen (+) or mock (−) inoculation areas. Observation at 24 hours post treatment. Mean values of 9 inoculations (3 inoculations on 3 plants by events). Bars indicate standard error of the 9 replicates.

(3) FIG. 3: Induction of chitinase gene promoter during P. pachyrhizi infection. Relative fluorescence intensity quantification of WT and Promoter-GFP (events 133 and 129) leaves on on pathogen (+) or mock (−) inoculated areas. Observations at 24, 48 and 72 hours post treatment. Mean values of 2 inoculations.

(4) The various aspects of the invention will be understood more fully by means of the experimental examples below.

(5) All the methods or operations described below are given by way of example and correspond to a choice, made among the various methods available for achieving the same result. This choice has no effect on the quality of the result, and, consequently, any appropriate method can be used by those skilled in the art to achieve the same result. In particular, and unless otherwise specified in the examples, all the recombinant DNA techniques employed are carried out according to the standard protocols described in Sambrook and Russel (2001, Molecular cloning: A laboratory manual, Third edition, Cold Spring Harbor Laboratory Press, NY) in Ausubel et al. (1994, Current Protocols in Molecular Biology, Current protocols, USA, Volumes 1 and 2), and in Brown (1998, Molecular Biology LabFax, Second edition, Academic Press, UK). Standard materials and methods for plant molecular biology are described in Croy R.D.D. (1993, Plant Molecular Biology LabFax, BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK)). Standard materials and methods for PCR (Polymerase Chain Reaction) are also described in Dieffenbach and Dveksler (1995, PCR Primer: A laboratory manual, Cold Spring Harbor Laboratory Press, NY) and in McPherson et al. (2000, PCR-Basics: From background to bench, First edition, Springer Verlag, Germany).

EXAMPLES

Example 1: Transcript Levels of the Chitinase Gene in Soybean after Phakopsora pachyrhizi Infection

(6) In order to measure transcript levels of the chitinase gene in soybean leaves, qPCR was used. Total RNA was extracted from soybean leaves by using the RNeasy® Plant Mini Kit (QIAGEN) and was treated with TURBO DNA-Free™ Kit (Invitrogen). 1 μg of RNA was used to synthetize cDNA with ThermoScript™ RT-PCR System kit (Invitrogen). The cDNA was diluted with 98 μL of RNAse free water to a final volume of 100 μL. 5 μL of diluted cDNA were used in a 20 μL reaction containing 10 μL of SsoAdvanced™ Universal SYBR® Grenn Supermix (BOI-RAD), 1 μL of primer forward, 1 μL of primer reverse and 3 μL of RNAse free water. qRT-PCR was performed using the LightCycler 480. Primers used to follow the expression of the chitinase gene are listed in Table 1. The thermocycling conditions were as follows: denaturation at 95° C. for 10 minutes and for amplification 45 cycles of 10 seconds at 95° C., 10 seconds at 60° C. and 10 seconds at 70° C. After the final cycle, the dissociation curve analysis was carried out to verify that the amplification occurred specifically and that no primer dimer product has been generated during the amplification process. The actin and metalloprotease genes (primer sequences in table 1) were used as endogenous reference genes to normalize the calculation using the Comparative Ct value method. The level of transcript abundance relative to the reference gene (termed ΔCt) was determined according to the function ΔCt=Ct (test gene)−Ct (reference gene). Then the function ΔΔCt was first determined using the equation ΔΔCt=ΔCt (treatment)−ΔCt (control) (where control represented mock-treated plants). The ratio of treatment/control was calculated by the equation 2{circumflex over ( )}(−ΔΔCt). All calculations have been realized with LightCycler® 480SW15 software.

(7) TABLE-US-00001 TABLE 1  Primer sequences for qRT-PCR Amplicon Gene Primer name Primer sequence length Actin GmACTIN_F CGGTGGTTCTATCTT 142 pb GGCATC (SEQ ID NO: 4) GmACTIN_R GTCTTTCGCTTCAAT AACCCTA (SEQ ID NO: 5) Metallo- Gmcons7_F* ATGAATGACGGTTCC 114 pb protease CATGTA (SEQ ID NO: 6) Gmcon7_R* GGCATTAAGGCAGCT CACTCT (SEQ ID NO: 7) Predicted  GmCHIT_F GAGATTAACGGTGCA 330 pb chitinase TCAGG (SEQ ID NO: 2) GmCHIT_R ATTAACACGAGCCTG AACAGTACT (SEQ ID NO: 3) *from Hirschburger et al., 2015

(8) Transcript levels were analyzed at 0 h, 8 h, 24 h, 48 h and 72 h post-infection by P. pachyrhizi. The results are reported in FIG. 1, showing significant increases in the levels of transcription of the gene from 8 h up to 72 h post-infection, thereby demonstrating the induction of the promoter of this gene following infection by P. pachyrhizi.

Example 2: Promoter Activity After Treatment with Various Elements

2.1. Construction of a Reporter Gene for Monitoring the Promoter Activity

(9) A DNA fragment of 3 454 bp upstream of the chitinase gene coding sequence, considered to comprise the putative promoter, was synthetized with addition of AatII and PlmII restriction enzyme sites respectively at the 5′ and 3′ end of the sequence. The putative promoter was cloned, using the restriction enzyme sites, to drive the expression of a GFP (Green-Fluorescent Protein) coding sequence in a vector also containing the gene coding for the HPPD enzyme (4-Hydroxyphenylpyruvate dioxygenase) under the control of a p35S promoter (used as selectable marker for plant transformation). The new vector obtained was named pBay00457. One positive clone was then sequenced from left border (LB) to right border (RB), and the T-DNA transferred to the plant via Agrobacterium tumefaciens transformation.

2.2. Soybean Treatment and Pathogens Inoculation

(10) For the phytohormone assays, two leaves of wild-type (WIT positive control or T1 plants transformed with the vector pBay00457 (containing 2 copies of the transgene) were harvested and placed on Watman filter with cotton around the petiole. One leaf was spread with chemicals inducer of phytophormone pathway (+) and one leaf was mock treated (−).

(11) For Jasmonate (JA) pathway activation, the leaves were sprayed with EC4% and methyl JA analogue (coronatine) at 3 ppm (+), or EC4% only (−).

(12) For Salicylate (SA) pathway activation, the leaves were treated with 2.5 mM of SA in Ethanol 10% (+), or Ethanol 10% only (−).

(13) For Ethylene (ET) pathway activation, the leaves were treated with the ethylene precursor (1-aminocyclopropane-1-carboxylic acid: ACC) at 20 mM (+) or H.sub.2O (−).

(14) Wounding was realized by cutting 3 leaf discs (0.3 cm of diameter) on a leave of WT plant, and on a leave of transformed “promoter-GFP” plant.

(15) Inoculations of P. pachyrhizi were realised via agar plugs (0.5 cm diameter). Cryo-conserved spores were first rehydrated 24 h before infection. 2 ml of a solution at 1 mg of spores/ml was equally spread on a plate of agar 3%, allowing a concentration of 600 spores/plug. Plugs were put on cut leaves which were incubated at 24° C., humidity 80% and 24 h in the dark, followed by a photoperiod 12/12. Plugs were removed 24 h post inoculation

(16) Inoculations of Sclerotinia sclerotiorum were realized with an agar plug of 5 days old fungus mycelium. Leaves were placed at 24° C., saturated humidity and photoperiod 12/12. Plugs were removed 48 hours post inoculation.

(17) Mock and pathogen inoculations were realized on the same leaf, with 3 mock inoculations on the left side and 3 inoculations with the fungus on the right side of the leaf.

2.3. GFP Observations

(18) The expression of green fluorescence rapporteur gene, GFP, under the control of the promoter according to the invention was measured in two soybean transformation events (events 133 and 129). Expression of the GFP was visualized with a macroscope camera LEICA Z16APO equipped with a GFP filter, lens 1×, magnification 6.95×, exposure time 1 s, gain 3. Fluorescence quantification was performed with MetaMorph software.

(19) For leaves infected by P. pachyrhizi, fluorescence observations were done at 0, 24, 48, and 72 hours post infection.

(20) For leaves infected by S. sclerotiorumn, fluorescence observations were done at 0, 48, and 72 hours post infection.

(21) For leaves either wounded or treated with the phytohormones, fluorescence observations were done at 0, 48, and 72 hours respectively post-wounding and post-treatment.

(22) A T2 homozygous soybean plant containing the GFP under the control of a promoter known to be responsive to biotic and abiotic stress (construct pBay00174, containing the promoter PDF1.2 from Arabidopsis, described in Manners et al. 1998, Plant Mol. Biol. 38: 1071-1080), was used as positive control. Results are shown in Table 2.

(23) TABLE-US-00002 TABLE 2 Expression profile of Promoter-GFP construct Biotic stress Abiotic Phytohormones Phakopsora Sclerotinia stress pathway induction Construct Event pachyrhizi sclerotiorum Wounding JA ET SA pBay00457 129 + − +.sup.(1) − − − 133 + nd +.sup.(1) − − − Ø WT − − − − − − pBay00174 Positive + + +.sup.(2) + + −.sup.(3) control Ø: absence of construct; −: no difference in GFP fluorescence observed after treatment; +: GFP fluorescence increase after treatment; nd: not determined .sup.(1)local response at the site of wounding only, no propagation .sup.(2)local and propagated response .sup.(3)the promoter used for the positive control (PDF1.2) is known not to be responsive to SA

(24) The results shown in Table 2 demonstrate that the promoter according to the invention is specifically induced by infection of the soybean plant with Phakopsora pachyrhizi, whereas it is not induced by the fungus Sclerotinia scierotiorum. These results also demonstrate that this promoter is not induced by most of the elements known to induce a defense response in plants, in particular a treatment with the phytohormones jasmonic acid, salicylic acid and ethylene. Finally, when it comes to the response to wounding, the results show that the promoter is activated locally at the point of wounding only.

(25) These experiments demonstrate the apparent specificity of this promoter to infection by P. pachyrhizi.

2.4. Promoter Expression Over Time after Infection by P. pachyrhizi

(26) The expression of GFP under the control of the promoter according to the invention was measured in two soybean transformation events (events 133 and 129) at 24 h, 48 h and 72 h post-infection by P. pachyrhizi. The results are reported in FIGS. 2 and 3.

(27) The results demonstrate the rapid induction of the promoter activity 24 h after infection by P. pachyrhizi (FIGS. 2 and 3), and its continuous activity over time at 48 h and 72 h post-infection (FIG. 3).

Example 3: Identification of Orthologous Promoters

(28) The fungal rust-inducible promoter according to the invention can be used to identify orthologous promoters, i.e. promoters having a substantially identical nucleic acid sequence and the same fungal rust-inducible functionality.

(29) This can, for example, be done, in silico, by looking into genomic databases for nucleic acid sequences having a substantially identical nucleic acid sequence in other plant species. For example, fungal rusts are known to infect many plant species, and therefore similar promoters with similar functionalities can be identified in such other species.

(30) Also, the Asian Soybean Rust, Phakopsora pachyrhizi, is known to infect other host plants than the soybean Glycine max, like e.g. Cajanus cajan, Lupinus sp., Phaseolus vulgaris or Vigna unquiculata, in which similar promoters with similar functionalities can also be identified.