Expanding agroinfiltration host range with broad-spectrum effectors monitored by autoluminescent reporter

Abstract

Agrobacterium-mediated transient transformation is a fundamental tool for plant research and molecular pharming, but its application is often limited by host immune responses, post-transcriptional gene silencing, and the lack of a robust, quantitative reporter for species beyond Nicotiana benthamiana. To address these challenges, we developed a sensitive reporter system based on the fungal bioluminescence pathway (FBP). As an autonomous, substrate-free metabolic pathway, the FBP enables high-throughput, quantitative screening without external manipulations. Using this system, we systematically evaluated factors affecting transformation efficiency across more than 20 plant families. This cross-lineage analysis identified that co-expressing NahG and P19, termed the NaP19 module, not only mitigates P19-induced cytotoxicity but also synergistically enhances transient expression in diverse species, including agroinfiltration-recalcitrant crops and horticultural plants. We demonstrate the platform’s in vivo utility in its native genetic background for applications, such as protein localization, interaction assays, and transcriptional regulation studies. By integrating a quantitative, substrate-free reporter with a broadly effective NaP19 enhancer, this work establishes a robust and versatile platform for advancing functional genomics and biotechnology throughout the land plant lineages.

References

[1] Yoo SD, Cho YH, Sheen J . Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc. 2007; 2: 1565-72

[2] Hsu CT, Chiu CC, Hsiao PY, et al. Transgene-free CRISPR-/Cas9-mediated gene editing through protoplast-to-plant regeneration enhances active compounds in. Plant Biotechnol J. 2024; 22: 1549-51

[3] Lomonossoff GP, D’Aoust MA . Plant-produced biopharmaceuticals: a case of technical developments driving clinical deployment. Science. 2016; 353: 1237-40

[4] Beritza K, Watts EC, van der Hoorn RAL . Improving transient protein expression in agroinfiltrated. New Phytol. 2024; 243: 846-50

[5] Murad S, Fuller S, Menary J, et al. Molecular pharming for low and middle income countries. Curr Opin Biotechnol. 2020; 61: 53-9

[6] Tschofen M, Knopp D, Hood E, et al. Plant molecular farming: much more than medicines. Annu Rev Anal Chem. 2016; 9: 271-94

[7] Ueki S, Lacroix B, Krichevsky A, et al. Functional transient genetic transformation of leaves by biolistic bombardment. Nat Protoc. 2009; 4: 71-7

[8] Rosas-Díaz T, Cana-Quijada P, Amorim-Silva V, et al. Plants as a suitable and efficient system for transient expression using. Mol Plant. 2017; 10: 353-6

[9] Zhang YJ, Chen M, Siemiatkowska B, et al. A highly efficient agrobacterium mediated method for transient gene expression and functional studies in multiple plant species. Plant Commun. 2020; 1: 100028

[10] Salazar-González JA, Castro-Medina M, Bernardino-Rivera LE, et al. In-planta transient transformation of avocado (Persea americana) by vacuum agroinfiltration of aerial plant parts. Plant Cell Tissue Org. 2023; 152: 635-46

[11] Zipfel C, Kunze G, Chinchilla D, et al. Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts mediated transformation. Cell. 2006; 125: 749-60

[12] Tsuda K, Qi Y, Nguyen LV, et al. An efficient agrobacterium-mediated transient transformation of Arabidopsis. Plant J. 2012; 69: 713-9

[13] Xie ZX, Fan BF, Chen CH, et al. An important role of an inducible RNA-dependent RNA polymerase in plant antiviral defense. Proc Natl Acad Sci USA. 2001; 98: 6516-21

[14] Yang SJ, Carter SA, Cole AB, et al. A natural variant of a host RNA-dependent RNA polymerase is associated with increased susceptibility to viruses by Nicotiana benthamiana. Proc Natl Acad Sci USA. 2004; 101: 6297-302

[15] Yuan ZC, Edlind MP, Liu P, et al. The plant signal salicylic acid shuts down expression of the regulon and activates quormone-quenching genes in. Proc Natl Acad Sci USA. 2007; 104: 11790-5

[16] Anand A, Uppalapati SR, Ryu CM, et al. Salicylic acid and systemic acquired resistance play a role in attenuating crown gall disease caused by Agrobacterium tumefaciens. Plant Physiol. 2008; 146: 703-15

[17] Rosas-Díaz T, Cana-Quijada P, Amorim-Silva V, et al. Plants as a suitable and efficient system for transient expression using Agrobacterium tumefaciens. Mol Plant. 2017; 10: 353-6

[18] Gopalakrishnan B, Wolff J . siRNA and DNA transfer to cultured cells. Methods Mol Biol. 2009; 480: 31-52

[19] Matsuo K, Matsumura T . Repression of the DCL2 and DCL4 genes in plants for the transient expression of recombinant proteins. J Biosci Bioeng. 2017; 124: 215-20

[20] Wu QF, Wang XB, Ding SW . Viral suppressors of RNA-based viral immunity: host targets. Cell Host Microbe. 2010; 8: 12-5

[21] Lombardi R, Circelli P, Villani ME, et al. High-level HIV-1 Nef transient expression in using the P19 gene silencing suppressor protein of artichoke mottled Crinckle virus. BMC Biotechnol. 2009; 9: 96

[22] Chudakov DM, Lukyanov S, Lukyanov KA . Fluorescent proteins as a toolkit for imaging. Trends Biotechnol. 2005; 23: 605-13

[23] Lippincott-Schwartz J, Patterson GH . Development and use of fluorescent protein markers in living cells. Science. 2003; 300: 87-91

[24] Yuan GL, Lu H, Tang D, et al. Expanding the application of a UV-visible reporter for transient gene expression and stable transformation in plants. Hortic Res. 2021; 8: 234

[25] Sun SY, Wang TE, Li YZ, et al. Advances in luminescence-based tracing technologies: applications and future perspectives. Biodesign Res. 2026; 8: 100060

[26] Sun SY, Ni Q, Luo D, et al. Development of an FBP-based bioluminescence reporter system for investigating transcriptional regulation in plants. Plant Commun. 2025; 6: 101143

[27] Koo J, Kim Y, Kim J, et al. A GUS/luciferase fusion reporter for plant gene trapping and for assay of promoter activity with luciferin-dependent control of the reporter protein stability. Plant Cell Physiol. 2007; 48: 1121-31

[28] He YB, Zhang T, Sun H, et al. A reporter for noninvasively monitoring gene expression and plant transformation. Hortic Res. 2020; 7: 152

[29] Kotlobay AA, Sarkisyan KS, Mokrushina YA, et al. Genetically encodable bioluminescent system from fungi. Proc Natl Acad Sci USA. 2018; 115: 12728-32

[30] Yu XL, Wang T, Kong C, et al. Engineering autonomously luminescent plants using fungal bioluminescence pathway. New Phytol. 2025; 248: 2251-61

[31] Zheng P, Ge J, Ji J, et al. Metabolic engineering and mechanical investigation of enhanced plant autoluminescence. Plant Biotechnol J. 2023; 21: 1671-81

[32] Mitiouchkina T, Mishin AS, Somermeyer LG, et al. Plants with genetically encoded autoluminescence. Nat Biotechnol. 2020; 38: 944-6

[33] Ge JY, Lang X, Ji J, et al. Integration of biological and information technologies to enhance plant autoluminescence. Plant Cell. 2024; 36: 4703-15

[34] Shakhova ES, Karataeva TA, Markina NM, et al. An improved pathway for autonomous bioluminescence imaging in eukaryotes. Nat Methods. 2024; 21: 406-10

[35] Calvache C, Vazquez-Vilar M, Moreno-Giménez E, et al. A quantitative autonomous bioluminescence reporter system with a wide dynamic range for plant synthetic biology. Plant Biotechnol J. 2024; 22: 37-47

[36] Khakhar A, Starker CG, Chamness JC, et al. Building customizable auto-luminescent luciferase-based reporters in plants. Elife. 2020; 9: e52786

[37] Rosas-Díaz T, Cana-Quijada P, Amorim-Silva V, et al. Arabidopsis NahG plants as a suitable and efficient system for transient expression using Agrobacterium tumefaciens. Mol Plant. 2017; 10: 353-6

[38] Oh CS, Martin GB . Effector-triggered immunity mediated by the Pto kinase. Trends Plant Sci. 2011; 16: 132-40

[39] Sarris PF, Gao S, Karademiris K, et al. Phytobacterial type III effectors HopX1, HopAB1 and HopF2 enhance sense-post-transcriptional gene silencing independently of plant R gene-effector recognition. Mol Plant Microbe In. 2011; 24: 907-17

[40] Saxena P, Hsieh YC, Alvarado VY, et al. Improved foreign gene expression in plants using a virus-encoded suppressor of RNA silencing modified to be developmentally harmless. Plant Biotechnol J. 2011; 9: 703-12

[41] Hamel LP, Tardif R, Poirier-Gravel F, et al. Molecular responses of agroinfiltrated Nicotiana benthamiana leaves expressing suppressor of silencing P19 and influenza virus-like particles. Plant Biotechnol J. 2024; 22: 1078-100

[42] Rueda EC, Dezar CA, Gonzalez DH, et al. Hahb-10, a sunflower homeobox-leucine zipper gene, is regulated by light quality and quantity, and promotes early flowering when expressed in Arabidopsis. Plant Cell Physiol. 2005; 46: 1954-63

[43] Dezar CA, Giacomelli JI, Manavella PA, et al. HAHB10, a sunflower HD-zip II transcription factor, participates in the induction of flowering and in the control of phytohormone-mediated responses to biotic stress. J Exp Bot. 2011; 62: 1061-76

[44] Yuan HM, Liu WC, Lu YT . CATALASE2 coordinates SA-mediated repression of both auxin accumulation and JA biosynthesis in plant defenses. Cell Host Microbe. 2017; 21: 143-55

[45] Lim ARQ, Kong Q, Singh SK, et al. Sunflower WRINKLED1 plays a key role in transcriptional regulation of oil biosynthesis. Int J Mol Sci. 2022; 23: 3054

[46] Li J, Nie K, Wang L, et al. The molecular mechanism of GhbHLH121 in response to iron deficiency in cotton seedlings. Plants-Basel. 2023; 12: 1955

[47] Zhang X, Wang L, Meng H, et al. Maize ABP9 enhances tolerance to multiple stresses in transgenic Arabidopsis by modulating ABA signaling and cellular levels of reactive oxygen species. Plant Mol Biol. 2011; 75: 365-78

[48] Wang CL, Lu G, Hao Y, et al. ABP9, a maize bZIP transcription factor, enhances tolerance to salt and drought in transgenic cotton. Planta. 2017; 246: 453-69

[49] Hamel L . Nicotiana benthamiana’s responses to agroinfiltration, a treasure grove of new avenues to improve protein yields in plant molecular farming. Plant Biotechnol J. 2026; 24: 5-17

[50] Pietrzykowska M, Suorsa M, Semchonok DA, et al. The light-harvesting chlorophyll a/b binding proteins Lhcb1 and Lhcb2 play complementary roles during state transitions in. Plant Cell. 2014; 26: 3646-60

[51] Jefferson RA . The Gus reporter gene system. Nature. 1989; 342: 837-8

[52] Cervera M . Histochemical and fluorometric assays for uidA (GUS) gene detection. Methods Mol Biol. 2005; 286: 203-14

[53] Baulcombe DC, Chapman S, Cruz SS . Jellyfish green fluorescent protein as a reporter for virus-infections. Plant J. 1995; 7: 1045-53

[54] Wang D, Zhong Y, Feng B, et al. The RUBY reporter enables efficient haploid identification in maize and tomato. Plant Biotechnol J. 2023; 21: 1707-15

[55] Sharifova S, Prasad KVSK, Cheema A, et al. Genetically encoded betalain-based RUBY visual reporters: noninvasive monitoring of biological processes. Trends Plant Sci. 2025; 30: 1332-45

[56] Pitzschke A. Infection and plant defense-transformation success hangs by a thread. Front Plant Sci. 2013; 4: 519

[57] Zhang J, Zhou JM . Plant immunity triggered by microbial molecular signatures. Mol Plant. 2010; 3: 783-93

[58] Raman V, Rojas CM, Vasudevan B, et al. Agrobacterium expressing a type III secretion system delivers Pseudomonas effectors into plant cells to enhance transformation. Nat Commun. 2022; 13: 2581

[59] Spoel SH, Dong XN . Salicylic acid in plant immunity and beyond. Plant Cell. 2024; 36: 1451-64

[60] Ngou BPM, Jones JDG, Ding PT . Plant immune networks. Trends Plant Sci. 2022; 27: 255-73

[61] Li Q, Liu Z, Jiang Z, et al. Phenylalanine metabolism-dependent lignification confers rhizobacterium-induced plant resistance. Plant Physiol. 2025; 197: kiaf016

[62] Hamilton A, Voinnet O, Chappell L, et al. Two classes of short interfering RNA in RNA silencing. EMBO J. 2002; 21: 4671-9

[63] Voinnet O, Lederer C, Baulcombe DC . A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana. Cell. 2000; 103: 157-67

[64] Reyna-Llorens I, Ferro-Costa M, Burgess SJ . Plant protoplasts in the age of synthetic biology. J Exp Bot. 2023; 74: 3821-32

[65] Jay F, Brioudes F, Voinnet O . A contemporary reassessment of the enhanced transient expression system based on the tombusviral silencing suppressor protein P19. Plant J. 2023; 113: 186-204

[66] Guo L, Yao H, Chen W, et al. Natural products of medicinal plants: biosynthesis and bioengineering in post-genomic era. Hortic Res. 2022; 9: uhac223

[67] Qin KZ, Liu F, Zhang C, et al. Systems and synthetic biology for plant natural product pathway elucidation. Cell Rep. 2025; 44: 115715

[68] Yu XL, Wei PL, Qu CY, et al. Sustainable bioenergy manufacturing in plants. Plant Commun. 2026; 7: 101711

[69] Zhu Q, Yu S, Zeng D, et al. Development of "purple endosperm rice" by engineering anthocyanin biosynthesis in the endosperm with a high-efficiency transgene stacking system. Mol Plant. 2017; 10: 918-29

[70] Barros J, Escamilla-Trevino L, Song L, et al. 4-Coumarate 3-hydroxylase in the lignin biosynthesis pathway is a cytosolic ascorbate peroxidase. Nat Commun. 2019; 10: 1994

[71] Marchler-Bauer A, Derbyshire MK, Gonzales NR, et al. CDD: NCBI’s conserved domain database. Nucleic Acids Res. 2015; 43: D222-6

[72] Katoh K, Standley DM . MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013; 30: 772-80

[73] Price TD . The roles of time and ecology in the continental radiation of the Old World leaf warblers (Phylloscopus and Seicercus). Philos Trans R Soc Lond Ser B Biol Sci. 2010; 365: 1749-62

[74] Tamura K, Stecher G, Kumar S . MEGA11 molecular evolutionary genetics analysis version 11. Mol Biol Evol. 2021; 38: 3022-7

[75] Lu J, Robert CAM, Riemann M, et al. Induced jasmonate signaling leads to contrasting effects on root damage and herbivore performance. Plant Physiol. 2015; 167: 1100-16

[76] Xi YX, Chochois V, Kroj T, et al. A novel robust and high-throughput method to measure cell death in leaves by fluorescence imaging. Mol Plant Pathol. 2021; 22: 1688-96