Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated Protein 9-generated Diallelic Mutants Reveal Arabidopsis Actin-related Protein 2 Function in the Trafficking of Syntaxin PEN1

Overview

Journal Front Plant Sci

Date 2022 Oct 7

PMID 36204067

Authors

Peng Gao

Li Qin

Hanh Nguyen

Huajin Sheng

Teagen D Quilichini

Daoquan Xiang

Leon V Kochian

Yangdou Wei

Raju Datla

Affiliations

Soon will be listed here.

Abstract

In plants, the actin cytoskeleton plays a critical role in defense against diverse pathogens. The formation of actin patches is essential for the intracellular transport of organelles and molecules toward pathogen penetration sites and the formation of papillae for an early cellular response to powdery mildew attack in . This response process is regulated by the actin-related protein (ARP)2/3 complex and its activator, the WAVE/SCAR complex (W/SRC). The ARP2/3 complex is also required for maintaining steady-state levels of the defense-associated protein, PENETRATION 1 (PEN1), at the plasma membrane and for its deposition into papillae. However, specific ARP2 functionalities in this context remain unresolved, as knockout mutants expressing reporter constructs could not be obtained by conventional crossing approaches. In this study, employing a CRISPR/Cas9 multiplexing-mediated genome editing approach, we produced an ARP2 knockout expressing the marker in . This study successfully identified diallelic somatic mutations with both alleles edited among the primary T1 transgenic plants, and also obtained independent lines with stable mutations in the T2 generation. Further analyses on these mutants showed similar biological functions of to in the accumulation of PEN1 against fungal invasion. Together, this CRISPR/Cas9-based approach offers highly efficient simultaneous disruption of the two alleles in -expressing lines, and a rapid method for performing live-cell imaging to facilitate the investigation of important plant-pathogen interactions using a well-established and widely applied GFP marker system, thus gaining insights and elucidating the contributions of ARP2 upon fungal attack.

References

Qin L, Liu L, Tu J, Yang G, Wang S, Quilichini T . The ARP2/3 complex, acting cooperatively with Class I formins, modulates penetration resistance in Arabidopsis against powdery mildew invasion. Plant Cell. 2021; 33(9):3151-3175. PMC: 8462814. DOI: 10.1093/plcell/koab170. View

Replogle J, Norman T, Xu A, Hussmann J, Chen J, Cogan J . Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing. Nat Biotechnol. 2020; 38(8):954-961. PMC: 7416462. DOI: 10.1038/s41587-020-0470-y. View

Pandey P, Quilichini T, Vaid N, Gao P, Xiang D, Datla R . Versatile and multifaceted CRISPR/Cas gene editing tool for plant research. Semin Cell Dev Biol. 2019; 96:107-114. DOI: 10.1016/j.semcdb.2019.04.012. View

Giacomello S, Salmen F, Terebieniec B, Vickovic S, Fernandez Navarro J, Alexeyenko A . Spatially resolved transcriptome profiling in model plant species. Nat Plants. 2017; 3:17061. DOI: 10.1038/nplants.2017.61. View

Hematy K, Lim M, Cherk C, Pislewska-Bednarek M, Sanchez-Rodriguez C, Stein M . Moonlighting Function of Phytochelatin Synthase1 in Extracellular Defense against Fungal Pathogens. Plant Physiol. 2020; 182(4):1920-1932. PMC: 7140922. DOI: 10.1104/pp.19.01393. View

cermak T, Curtin S, Gil-Humanes J, Cegan R, Kono T, Konecna E . A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants. Plant Cell. 2017; 29(6):1196-1217. PMC: 5502448. DOI: 10.1105/tpc.16.00922. View

Nolen B, Pollard T . Structure and biochemical properties of fission yeast Arp2/3 complex lacking the Arp2 subunit. J Biol Chem. 2008; 283(39):26490-8. PMC: 2546537. DOI: 10.1074/jbc.M802607200. View

Thordal-Christensen H . Fresh insights into processes of nonhost resistance. Curr Opin Plant Biol. 2003; 6(4):351-7. DOI: 10.1016/s1369-5266(03)00063-3. View

Wang Z, Xing H, Dong L, Zhang H, Han C, Wang X . Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biol. 2015; 16:144. PMC: 4507317. DOI: 10.1186/s13059-015-0715-0. View

10.

Yang L, Qin L, Liu G, Peremyslov V, Dolja V, Wei Y . Myosins XI modulate host cellular responses and penetration resistance to fungal pathogens. Proc Natl Acad Sci U S A. 2014; 111(38):13996-4001. PMC: 4183272. DOI: 10.1073/pnas.1405292111. View

11.

Assaad F, Qiu J, Youngs H, Ehrhardt D, Zimmerli L, Kalde M . The PEN1 syntaxin defines a novel cellular compartment upon fungal attack and is required for the timely assembly of papillae. Mol Biol Cell. 2004; 15(11):5118-29. PMC: 524786. DOI: 10.1091/mbc.e04-02-0140. View

12.

Yun B, Atkinson H, Gaborit C, Greenland A, Read N, Pallas J . Loss of actin cytoskeletal function and EDS1 activity, in combination, severely compromises non-host resistance in Arabidopsis against wheat powdery mildew. Plant J. 2003; 34(6):768-77. DOI: 10.1046/j.1365-313x.2003.01773.x. View

13.

Wiedenheft B, Sternberg S, Doudna J . RNA-guided genetic silencing systems in bacteria and archaea. Nature. 2012; 482(7385):331-8. DOI: 10.1038/nature10886. View

14.

Szymanski D . Breaking the WAVE complex: the point of Arabidopsis trichomes. Curr Opin Plant Biol. 2005; 8(1):103-12. DOI: 10.1016/j.pbi.2004.11.004. View

15.

Underwood W, Somerville S . Perception of conserved pathogen elicitors at the plasma membrane leads to relocalization of the Arabidopsis PEN3 transporter. Proc Natl Acad Sci U S A. 2013; 110(30):12492-7. PMC: 3725069. DOI: 10.1073/pnas.1218701110. View

16.

Brooks C, Nekrasov V, Lippman Z, Van Eck J . Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system. Plant Physiol. 2014; 166(3):1292-7. PMC: 4226363. DOI: 10.1104/pp.114.247577. View

17.

Takemoto D, Jones D, Hardham A . GFP-tagging of cell components reveals the dynamics of subcellular re-organization in response to infection of Arabidopsis by oomycete pathogens. Plant J. 2003; 33(4):775-92. DOI: 10.1046/j.1365-313x.2003.01673.x. View

18.

Moseley J, Goode B . The yeast actin cytoskeleton: from cellular function to biochemical mechanism. Microbiol Mol Biol Rev. 2006; 70(3):605-45. PMC: 1594590. DOI: 10.1128/MMBR.00013-06. View

19.

Pollard T . Regulation of actin filament assembly by Arp2/3 complex and formins. Annu Rev Biophys Biomol Struct. 2007; 36:451-77. DOI: 10.1146/annurev.biophys.35.040405.101936. View

20.

Kuhn H, Kwaaitaal M, Kusch S, Acevedo-Garcia J, Wu H, Panstruga R . Biotrophy at Its Best: Novel Findings and Unsolved Mysteries of the Arabidopsis-Powdery Mildew Pathosystem. Arabidopsis Book. 2016; 14:e0184. PMC: 4957506. DOI: 10.1199/tab.0184. View