Agrobacterium-mediated transient transformation of sorghum leaves for accelerating functional genomics and genome editing studies.

doi:10.21203/rs.2.11984/v1

PDF

Research note

Agrobacterium-mediated transient transformation of sorghum leaves for accelerating functional genomics and genome editing studies.

https://doi.org/10.21203/rs.2.11984/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 27 Feb, 2020

Read the published version in BMC Research Notes →

Version 1

posted 26 Jul, 2019

You are reading this latest preprint version

Objectives Sorghum is one of the most recalcitrant species for transformation. Considering the time and effort required for stable transformation in sorghum, establishing a transient system to screen the efficiency and full functionality of vector constructs is highly desirable. Results Here, we report an Agrobacterium-mediated transient transformation assay with intact sorghum leaves using green fluorescent protein as marker. It also provides a good monocot alternative to tobacco and protoplast assays with a direct, native and more reliable system for testing single guide RNA (sgRNA) expression construct efficiency. Given the simplicity and ease of transformation, high reproducibility, and ability to test large constructs, this method can be widely adopted to speed up functional genomic and genome editing studies.

Plant Molecular Biology and Genetics

Agrobacterium

CRISPR

sgRNA

sorghum

transformation

transient

Sorghum is a gluten-free C4 crop, important as both a human dietary staple and animal feed, but more recently also as a potential feedstock for biofuel production[1]. With high collinearity and synteny with other grass genomes, sorghum also provides an ideal template to serve as model for other grasses[2]. However, realizing the full potential of sorghum as feedstock requires bioengineering efforts aimed at tailoring sorghum biomass for biorefining applications [3, 4]. Indeed, while the sorghum genome sequence was completed a decade ago [2], only a handful of genes have been characterized using transgenic approaches.

A major factor in the lack of progress is the low efficiency and time-consuming nature of stable transformation. Indeed, sorghum is one of the most recalcitrant crops to transformation and regeneration. The first sorghum transgenic plants were generated using particle bombardment in 1993 with only 0.28% transformation rate[5]. Subsequently, Zhao and coworkers[6] reported 2.12% transformation rate using Agrobacterium-mediated transformation. Although with recent advancements in technology and optimization of regeneration protocols, several labs have been able to now transform a few limited sorghum cultivars with improved efficiency; reproducibility and consistency still remain major issues[7–9].

When developing engineered plants, due to the time and cost involved, it is highly desirable to test construct functionality in a transient assay. This is particularly true for sorghum. Transient assays in grasses mostly rely on protoplasts[12–14]. However, expression of a gene in protoplasts may not always mimic in planta native state and, also experience inconsistent efficiency due to variability in quality of protoplasts and size of vector transformed[15]. Here, we have established a simplified transient assay with Agrobacterium, also known as agroinfiltration, for transient transformation of sorghum and demonstrated its application by confirming gene editing in sorghum leaves using GFP as a marker. Using our method, researchers can directly test the in planta efficacy of binary constructs that may subsequently be used for stable transformation.

Plasmids and bacterial strains

Binary vectors C282 and C283 were built based on pTKan-p35S-attR1-GW-attR2 backbone vector[16] using Gateway{Gonzalez, 2015 #3120} (Invitrogen, CA, U.S. A.) to introduce codons for GFP (C282) or frame-shifted (fs)GFP (C283) for expression under the CaMV 35S promoter. The fsGFP has a 23 bp positive target control (PTC) sequence inserted after the start codon (5’-gcgcttcaaggtgcacatggagg–3’)[21]. C286 contains GFP driven by maize Ubiquitin 1 promoter, described elsewhere[17, 18]. Binary vectors C475 and C476 were built based on pTKan-pNOS-DsRed-pZmUBQ1-attR1-GW-attR2 backbone vector[17]. The C476 cassette (pTKan-pNOS-DsRed-tNOS-pZmUBQ1-CAS9p-pOsU3-PTC_gRNA-p35S-fsGFP) contains a sgRNA (5’-gcgcttcaaggtgcacatgg–3’) targeting the PTC sequence in fsGFP. CAS9p is a plant codon optimized CAS9 from Streptococcus pyogenes[19]. The C475 cassette (pTKan-pNOS-DsRed-tNOS-pZmUBQ1-CAS9p-pOsU3-nongRNA-p35S-fsGFP) lacking a sgRNA targeting sequence was used as a negative control. Plasmids are available from the JBEI registry: https://registry.jbei.org

Binary vectors were transformed into Agrobacterium tumefaciens strain GV3101 using electroporation, and grown in Luria Bertani (LB) medium containing 100/30/50 g/mL rifampicin/gentamicin/spectinomycin at 28C. Similarly, A. tumefaciens strain C58C1 containing the P19 suppressor of gene-silencing protein was grown in LB media containing 100/5/50 g/mL rifampicin/tetracycline/kanamycin.

Leaf infiltration

For agroinfiltration, Agrobacterium was grown in liquid culture (5 mL, 24 h, 30C), and cells were pelleted (5000 xg, 5 min), and resuspended in infiltration medium containing 50 mM MES, pH 5.6, 2 mM Na₃PO₄, 0.5% (w/v) dextrose, 200 M acetosyringone and 0.01% Silwet L–77 with an OD₆₀₀of 0.5. The P19 strain was mixed with each of the other strains to ¼ of the final volume. Prior to infiltration, the Agrobacterium suspension was incubated without shaking at 30C for about 2 h. The Nicotiana benthamiana plants were grown in a growth chamber under 16/8 h and 26/24C day/night cycle, and plants of ~4-weeks-old used for infiltration. Sorghum bicolor (L.) Moench inbred line Tx430 plants were grown in a plant growth room under 14/10 h 29/26C day/night cycle. Plants at the three-leaf stage (3–4 weeks old), were used for co-infiltration (Fig. 1). The fully expanded sorghum leaves were mechanically wounded with a syringe needle (0.8 mm X 40 mm) several times to make the epidermis more conducive to infiltration. No injury was caused to tobacco leaves. The Agrobacterium strains suspended in infiltration medium were infiltrated into leaves using a 1 mL syringe without needle. The boundaries of regions infiltrated with Agrobacterium were marked with a permanent marker for later visualization.

Microscopy

About 3–4 days after infiltration (DAI), tobacco and sorghum leaves were detached from the plant and observed under a Leica D4000B fluorescence microscope coupled with a Leica DC500 camera using appropriate filters for GFP and DsRed.

Expression of GFP in infiltrated leaves of tobacco and sorghum

We tested binary constructs C282 containing 35S_pro::GFP and the modified plasmid C283 with 35S_pro::fsGFP (frame-shifted GFP) by agroinfiltration in both tobacco and sorghum leaves. At 3DAI, the GFP signal was examined in detached leaves under a fluorescent microscope. Both sorghum and tobacco leaves infiltrated with C282 showed high and consistent expression of GFP (Fig. 2). However, those infiltrated with C283, containing fsGFP, exhibited no signal. It was noted that the area of detectable GFP expression was much smaller in sorghum as compared to tobacco. This is likely due to the limited infiltration of Agrobacterium suspension in sorghum leaves. The signal could be observed up to 7 DAI, after which the signal declined.

Ubiquitin promoter is more effective for sorghum

We compared infiltration of plasmid C282 (35S_pro::GFP) with C286 (Ubq_pro::GFP) in sorghum. While a higher intensity of GFP signal was observed in tobacco leaves with the 35S promoter compared to sorghum leaves (Fig. 2); GFP expression driven by the maize ubiquitin1 promoter exhibited higher intensity in sorghum leaves.

Demonstration of gene editing in sorghum leaves using GFP as target gene

To test whether we can use our transient Agrobacterium-mediated transformation method to determine sgRNA gene editing efficiency in sorghum, we used the binary vectors, C475 and C476 for agroinfiltration. Tobacco leaves were also infiltrated as a comparison. Both C475 and C476 contained constitutively expressed DsRed under NOS promoter, fsGFP driven by 35S promoter and pUbi-driven CAS9p for CRISPR-mediated genome editing. C476 contained a sgRNA targeting the PTC sequence in fsGFP. As C475 lacked the targeting sgRNA, GFP expression was only expected with C476 vector and only when editing occurs to correct the GFP frame shift.

Following agroinfiltraion, DsRed expression could be detected in both sorghum and tobacco leaves with both the constructs, confirming successful infiltration (Fig. 3). However, GFP expression was observed only in the leaves infiltrated with C476 demonstrating successful editing in the intact leaves of both tobacco and sorghum (Fig. 3).

Plant transformation is indispensable for elucidating gene function and engineering plant genomes for improved agronomic traits. Several biological, mechanical, chemical and electrical methods of DNA delivery have been developed to facilitate plant transformation over past several decades[22, 23]. Among biological methods, the soil-borne gram-negative bacterium A. tumefaciens is no doubt the most popular and widely used vehicle for DNA delivery in plant cells[24]. Although monocots are outside the host range of this bacterium, Agrobacterium-mediated transformation is now routinely used for transforming monocot genomes as well, though with lower efficiency[25, 26]. Agroinfiltration is also routinely used in several plant species due to rapidity, versatility and convenience[27–33]. However, success of this method in monocot species is very limited primarily due to extensive epidermal cuticular wax, high silica content, and low volume of intercellular space. These morphological features prevent the infiltration of bacterial cells into grasses via the application of simple pressure. Although microprojectile bombardment may be used to introduce expression constructs in cereals, the set-up cost for establishing microprojectile bombardment is high. Moreover, it only targets single cells limiting the scope of screening[34], and often leads to cell damage. Earlier, Andrieu and coworkers[35] reported Agrobacterium-mediated transient gene expression and silencing in rice leaves by mechanically wounding leaves followed by direct incubation in Agrobacterium suspension. However, we made several attempts to transform sorghum leaves at different stages of development, using their methodology, but could not detect any expression of GFP (data not shown).

Virus-based vectors provide an alternative opportunity for elucidating monocot gene functions. However, instability of the recombinant vector, improper orientation of insert and inconsistency due to inadequate infectivity, inoculation methods, replication/movement of virus in the host, pose serious challenges[36]. Another recent study demonstrated application of nanoparticles in transformation of wheat leaves by combining wounding treatment with syringe infiltration of the nanoparticles[37]. However, the size of plasmid that can be loaded onto nanoparticles is a major constraint due to size exclusion limit of the plant cell wall (~20 nm).

To overcome these constraints, we attempted syringe infiltration with recombinant Agrobacterium, containing vectors for in planta GFP expression, at different stages of development in sorghum leaves. As expected, strength of signal in sorghum leaves was higher with the maize ubiquitin promoter as compared to cauliflower mosaic virus 35S promoter, which is reported to perform better in dicots[38]. In our system, although infiltration medium could enter the mature leaves, GFP expression was only detected in the infiltrated younger leaves of 3–4-week-old plants. The expression of GFP seemed to localize to where bacteria were initially infiltrated through mechanical pressure. We did not observe a spread of signal in the adjacent areas, unlike that reported by Andrieu and coworkers[35] for siRNAs in rice. This observation indicated that although bacteria could enter sorghum leaf cells through the wounded regions, they could not passively diffuse to other cells without mechanical pressure in sorghum leaves. We also tried dipping the leaf in Agrobacterium suspension after clipping the leaf from the top, as well as wounding by needle, however Agrobacterium could not detectably enter the sorghum leaves without applied mechanical pressure.

Further, we demonstrated the application of our method to test the efficiency of sgRNA in genome editing constructs. CRISPR-associated Cas9 is a powerful genome editing tool for engineering plants[39]. Although the design of sgRNAs and preparation of constructs is straightforward, the accuracy and efficiency of the method relies on the choice of sgRNAs[40]. Several in silico prediction tools are available to predict the efficiency of sgRNAs based on the sequence features. However, predicted sgRNAs often have vastly different editing efficiencies in planta[18]. Protoplasts have been commonly used to test sgRNA efficiency. However, obtaining high quality protoplasts for genome editing needs extensive standardization, especially for plants such as sorghum. Secondly, additional cloning steps have to be performed to obtain a smaller vector for protoplast transformation. Thirdly and most importantly, the efficiency predicted in protoplasts may not correlate with the efficiency observed in intact plant tissue[40]. Therefore, screening of sgRNAs to achieve high accuracy and efficiency remains a challenge. We adopted our Agrobacterium-mediated transient transformation strategy to test sgRNA-mediated editing efficiency in sorghum leaves. The editing was observed in the transformed tissue within three days after infiltration, thereby providing a reliable assay for testing sgRNAs under native conditions.

We used GFP as a reporter in our study as it allows direct visualization in living tissues without being invasive or destructive and does not need any substrate. Gao and workers[41] demonstrated successful use of GFP as marker for stable transformation in sorghum, avoiding use of antibiotics or herbicides. This strategy can be easily applied in our system to quickly assess the full functionality of the vector constructs. For sgRNAs targeting endogenous genes, efficacy can be tested using RT-PCR or sequencing.

Overall, our study demonstrated that in planta Agrobacterium-mediated transient expression of transgenes is achievable in sorghum leaves. High reproducibility, simplicity, rapidity and feasibility to transform large constructs, which can directly be used for stable transformation, are the key advantages of our method. Though this method can be used for subcellular localization studies and physiological assays, the ability to test sgRNA targeting efficiency should be of particular interest.

The efficiency of agroinfiltration is much less compared to that observed in tobacco plants and therefore infiltration of more plants may be necessary if significant amount of materials are required for downstream analysis.
Since we were targeting a transgene in our editing assays, editing of an endogenous sorghum gene and confirmation of successful editing by sequencing would be an important step to confirm wide applicability of this method.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and material

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests

Funding

R. S. acknowledges IUSSTF-DBT for GETin fellowship. This work was funded by DOE Joint BioEnergy Institute (http://www.jbei.org) supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research through Contract DEAC0205CH11231 between Lawrence Berkeley National Laboratory and the U.S. Department of Energy.

Author’s Contributions

RS, JCM and HVS conceptualized the study and designed the experiments. RS, YL, VRP and MYL performed the experiments and analyzed the data. RS drafted the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable

1.Mathur S, Umakanth AV, Tonapi VA, Sharma R, Sharma MK: Sweet sorghum as biofuel feedstock: recent advances and available resources. Biotechnol Biofuels 2017, 10:146.

2.Paterson AH, Bowers JE, Bruggmann R, Dubchak I, Grimwood J, Gundlach H, Haberer G, Hellsten U, Mitros T, Poliakov A et al: The Sorghum bicolor genome and the diversification of grasses. Nature 2009, 457(7229):551–556.

3.van der Weijde T, Alvim Kamei CL, Torres AF, Vermerris W, Dolstra O, Visser RG, Trindade LM: The potential of C4 grasses for cellulosic biofuel production. Front Plant Sci 2013, 4:107.

4.Bhatia R, Gallagher JA, Gomez LD, Bosch M: Genetic engineering of grass cell wall polysaccharides for biorefining. Plant Biotechnol J 2017, 15(9):1071–1092.

5.Casas AM, Kononowicz AK, Zehr UB, Tomes DT, Axtell JD, Butler LG, Bressan RA, Hasegawa PM: Transgenic sorghum plants via microprojectile bombardment. Proc Natl Acad Sci U S A 1993, 90(23):11212–11216.

6.Zhao ZY, Cai T, Tagliani L, Miller M, Wang N, Pang H, Rudert M, Schroeder S, Hondred D, Seltzer J et al: Agrobacterium-mediated sorghum transformation. Plant Mol Biol 2000, 44(6):789–798.

7.Ahmed RI, Ding A, Xie M, Kong Y: Progress in Optimization of Agrobacterium-Mediated Transformation in Sorghum (Sorghum bicolor). Int J Mol Sci 2018, 19(10).

8.Che P, Anand A, Wu E, Sander JD, Simon MK, Zhu W, Sigmund AL, Zastrow-Hayes G, Miller M, Liu D et al: Developing a flexible, high-efficiency Agrobacterium-mediated sorghum transformation system with broad application. Plant Biotechnol J 2018, 16(7):1388–1395.

9.Nelson-Vasilchik K, Hague J, Mookkan M, Zhang ZJ, Kausch A: Transformation of Recalcitrant Sorghum Varieties Facilitated by Baby Boom and Wuschel2. Curr Protoc Plant Biol 2018, 3(4):e20076.

10.Girijashankar V, Swathisree V: Genetic transformation of Sorghum bicolor. Physiol Mol Biol Plants 2009, 15(4):287–302.

11.Jeoung JM, Krishnaveni S, Muthukrishnan S, Trick HN, Liang GH: Optimization of sorghum transformation parameters using genes for green fluorescent protein and beta-glucuronidase as visual markers. Hereditas 2002, 137(1):20–28.

12.Burris KP, Dlugosz EM, Collins AG, Stewart CN, Jr., Lenaghan SC: Development of a rapid, low-cost protoplast transfection system for switchgrass (Panicum virgatum L.). Plant Cell Rep 2016, 35(3):693–704.

13.Lin CS, Hsu CT, Yang LH, Lee LY, Fu JY, Cheng QW, Wu FH, Hsiao HC, Zhang Y, Zhang R et al: Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration. Plant Biotechnol J 2018, 16(7):1295–1310.

14.Zhang Y, Su J, Duan S, Ao Y, Dai J, Liu J, Wang P, Li Y, Liu B, Feng D et al: A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes. Plant Methods 2011, 7(1):30.

15.Rehman L, Su X, Guo H, Qi X, Cheng H: Protoplast transformation as a potential platform for exploring gene function in Verticillium dahliae. BMC Biotechnol 2016, 16(1):57.

16.Gonzalez TL, Liang Y, Nguyen BN, Staskawicz BJ, Loque D, Hammond MC: Tight regulation of plant immune responses by combining promoter and suicide exon elements. Nucleic Acids Res 2015, 43(14):7152–7161.

17.Liang Y, Richardson S, Yan J, Benites VT, Cheng-Yue C, Tran T, Mortimer J, Mukhopadhyay A, Keasling JD, Scheller HV et al: Endoribonuclease-Based Two-Component Repressor Systems for Tight Gene Expression Control in Plants. ACS Synth Biol 2017, 6(5):806–816.

18.Liang Y, Eudes A, Yogiswara S, Jing B, Benites VT, Yamanaka R, Cheng-Yue C, Baidoo EE, Mortimer JC, Scheller HV et al: A screening method to identify efficient sgRNAs in Arabidopsis, used in conjunction with cell-specific lignin reduction. Biotechnol Biofuels 2019, 12:130.

19.Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y et al: A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot Plants. Molecular plant 2015, 8(8):1274–1284.

20.Sparkes IA, Runions J, Kearns A, Hawes C: Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc 2006, 1(4):2019–2025.

21.Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP: Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res 2013.

22.Altpeter F, Springer NM, Bartley LE, Blechl AE, Brutnell TP, Citovsky V, Conrad LJ, Gelvin SB, Jackson DP, Kausch AP et al: Advancing Crop Transformation in the Era of Genome Editing. Plant Cell 2016, 28(7):1510–1520.

23.Barampuram S, Zhang ZJ: Recent advances in plant transformation. Methods Mol Biol 2011, 701:1–35.

24.Gelvin SB: Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol Mol Biol Rev 2003, 67(1):16–37, table of contents.

25.Wu E, Zhao ZY: Agrobacterium-Mediated Sorghum Transformation. Methods Mol Biol 2017, 1669:355–364.

26.Hiei Y, Ishida Y, Komari T: Progress of cereal transformation technology mediated by Agrobacterium tumefaciens. Front Plant Sci 2014, 5:628.

27.Bhaskar PB, Venkateshwaran M, Wu L, Ane JM, Jiang J: Agrobacterium-mediated transient gene expression and silencing: a rapid tool for functional gene assay in potato. PLoS One 2009, 4(6):e5812.

28.Circelli P, Donini M, Villani ME, Benvenuto E, Marusic C: Efficient Agrobacterium-based transient expression system for the production of biopharmaceuticals in plants. Bioeng Bugs 2010, 1(3):221–224.

29.Figueiredo JF, Romer P, Lahaye T, Graham JH, White FF, Jones JB: Agrobacterium-mediated transient expression in citrus leaves: a rapid tool for gene expression and functional gene assay. Plant Cell Rep 2011, 30(7):1339–1345.

30.Kim MJ, Baek K, Park CM: Optimization of conditions for transient Agrobacterium-mediated gene expression assays in Arabidopsis. Plant Cell Rep 2009, 28(8):1159–1167.

31.Krenek P, Samajova O, Luptovciak I, Doskocilova A, Komis G, Samaj J: Transient plant transformation mediated by Agrobacterium tumefaciens: Principles, methods and applications. Biotechnol Adv 2015, 33(6 Pt 2):1024–1042.

32.Li JF, Nebenfuhr A: FAST technique for Agrobacterium-mediated transient gene expression in seedlings of Arabidopsis and other plant species. Cold Spring Harb Protoc 2010, 2010(5):pdb prot5428.

33.Zheng L, Liu G, Meng X, Li Y, Wang Y: A versatile Agrobacterium-mediated transient gene expression system for herbaceous plants and trees. Biochem Genet 2012, 50(9–10):761–769.

34.Schweizer P, Pokorny J, Schulze-Lefert P, Dudler R: Technical advance. Double-stranded RNA interferes with gene function at the single-cell level in cereals. Plant J 2000, 24(6):895–903.

35.Andrieu A, Breitler JC, Sire C, Meynard D, Gantet P, Guiderdoni E: An in planta, Agrobacterium-mediated transient gene expression method for inducing gene silencing in rice (Oryza sativa L.) leaves. Rice (N Y) 2012, 5(1):23.

36.Kant R, Dasgupta I: Gene silencing approaches through virus-based vectors: speeding up functional genomics in monocots. Plant Mol Biol 2019.

37.Demirer GS, Zhang H, Matos JL, Goh NS, Cunningham FJ, Sung Y, Chang R, Aditham AJ, Chio L, Cho MJ et al: High aspect ratio nanomaterials enable delivery of functional genetic material without DNA integration in mature plants. Nat Nanotechnol 2019.

38.Somssich M: A Short History of the CaMV 35S Promoter. PeerJ PrePr 2018, 6(e27096v2):1–16.

39.Khatodia S, Bhatotia K, Passricha N, Khurana SM, Tuteja N: The CRISPR/Cas Genome-Editing Tool: Application in Improvement of Crops. Front Plant Sci 2016, 7:506.

40.Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I, Sullender M, Ebert BL, Xavier RJ, Root DE: Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol 2014, 32(12):1262–1267.

41.Gao Z, Jayaraj J, Muthukrishnan S, Claflin L, Liang GH: Efficient genetic transformation of Sorghum using a visual screening marker. Genome 2005, 48(2):321–333.

PDF

Journal Publication

published 27 Feb, 2020

Read the published version in BMC Research Notes →

Version 1

posted 26 Jul, 2019

You are reading this latest preprint version

Agrobacterium-mediated transient transformation of sorghum leaves for accelerating functional genomics and genome editing studies.

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Methods

Plasmids and bacterial strains

Leaf infiltration

Microscopy

Results

Expression of GFP in infiltrated leaves of tobacco and sorghum

Ubiquitin promoter is more effective for sorghum

Demonstration of gene editing in sorghum leaves using GFP as target gene

Discussion

Limitations

Declarations

Ethics approval and consent to participate

Consent for publication

Availability of data and material

Competing interests

Funding

Author’s Contributions

Acknowledgements

References

Status:

Journal Publication

Version 1