Floral Stage Optimization and Immune Evasion Enhance Agrobacterium-Mediated Genome Editing in Arabidopsis

Floral Stage Optimization and Immune Evasion Enhance Agrobacterium-Mediated Genome Editing in Arabidopsis | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Floral Stage Optimization and Immune Evasion Enhance Agrobacterium -Mediated Genome Editing in Arabidopsis View ORCID Profile Mao-Sen Liu , View ORCID Profile Teng-Kuei Huang , Yi-Chieh Wang , View ORCID Profile Si-Chong Wang , View ORCID Profile Chih-Hang Wu , View ORCID Profile Chih-Horng Kuo , View ORCID Profile Erh-Min Lai doi: https://doi.org/10.1101/2025.05.07.652770 Mao-Sen Liu 1 Institute of Plant and Microbial Biology , Academia Sinica, Taipei, Taiwan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mao-Sen Liu Teng-Kuei Huang 1 Institute of Plant and Microbial Biology , Academia Sinica, Taipei, Taiwan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Teng-Kuei Huang Yi-Chieh Wang 1 Institute of Plant and Microbial Biology , Academia Sinica, Taipei, Taiwan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Si-Chong Wang 1 Institute of Plant and Microbial Biology , Academia Sinica, Taipei, Taiwan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Si-Chong Wang Chih-Hang Wu 1 Institute of Plant and Microbial Biology , Academia Sinica, Taipei, Taiwan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Chih-Hang Wu Chih-Horng Kuo 1 Institute of Plant and Microbial Biology , Academia Sinica, Taipei, Taiwan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Chih-Horng Kuo Erh-Min Lai 1 Institute of Plant and Microbial Biology , Academia Sinica, Taipei, Taiwan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Erh-Min Lai For correspondence: emlai{at}gate.sinica.edu.tw Abstract Full Text Info/History Metrics Preview PDF SUMMARY Agrobacterium -mediated transformation via floral inoculation (AMT-FI) enables genetic engineering without tissue culture. It is widely used in the model plant Arabidopsis thaliana , yet its efficiency and broader applicability remain limited. Here, we used a dual-reporter system (RUBY and hygromycin resistance) to identify key floral stages and engineered Agrobacterium strains to evade plant immunity, leading to enhanced transient expression and genome editing. We determined that flowers opened at 6 day-post-inoculation (DPI) are optimal for high transformation efficiency, with nearly 100% of siliques harboring transformants. However, Agrobacterium infection induced ovule abortion, particularly in wild-type (Col-0) plants, whereas efr mutants lacking the EF-Tu receptor (EFR)-mediated pattern-triggered immunity (PTI) showed reduced ovule abortion. Notably, efr mutants exhibited more RUBY-positive ovules and significantly enhanced genome editing efficiency. Two engineered stealth Agrobacterium strains (AS201 and AS202) expressing a chimeric EF- Tu for evading recognition by EFR enhanced both transient transformation and genome editing efficiency. Remarkably, genome-edited T1 plants could be recovered based on phenotype or direct sequencing without the need for antibiotic selection when targeting flowers opened at 6 DPI. By integrating floral stage selection, immune evasion, and Agrobacterium engineering, this study provides a practical and versatile platform to advance plant genome engineering. INTRODUCTION Agrobacterium species are unique bacteria capable of cross-kingdom gene transfer, leading to diseases like crown gall or hairy root in a wide range of plant species ( Hwang et al ., 2017 ; Lacroix & Citovsky, 2019 ). This remarkable ability has been harnessed in plant genetic engineering by replacing the oncogenes within T-DNA region of tumor-inducing plasmids with genes of interest ( Suzuki et al ., 2009 ; Hooykaas, 2023 ). Agrobacterium- mediated transformation (AMT) has become a cornerstone technique in plant biology and the development of genetically modified organisms (GMOs) that stably express agricultural traits for crop improvements ( Bevan, 1984 ; Gelvin, 2003 ). Beyond enabling stable gene integration, AMT also supports transient expression of genome-editing machinery, allowing precise genome modifications. Since the T-DNA containing these editing components can be eliminated via chromosome segregation in subsequent generations, this strategy mitigates food safety concerns associated with GMOs ( Pixley et al ., 2022 ). Agrobacterium naturally infects somatic plant cells by responding to wound signals in roots or stems. Interestingly, it can also target germline tissues such as female gametophytes, which laid the foundation for the floral dip method widely used in Arabidopsis thaliana ( Clough & Bent, 1998 ; Angulo et al ., 2023 ). This method, referred to here as Agrobacterium- mediated transformation by floral inoculation (AMT-FI), bypasses labor-intensive tissue culture, offering a more efficient transformation pathway. However, its application in species beyond A. thaliana has limited success ( Bélanger et al ., 2024 ). Although many plant factors that promote or suppress tumorigenesis and AMT have been identified, the genes involved in AMT-FI may be distinct ( Hwang et al ., 2017 ; Lacroix & Citovsky, 2019 ; Weisberg et al ., 2023 ). Several Arabidopsis mutants or ecotypes resistant to root transformation remain susceptible to AMT-FI ( Nam et al ., 1997 ; Mysore et al ., 2000 ). Notably, polQ mutants lacking DNA polymerase theta are deficient in T-DNA integration via AMT-FI, although their capability for stable transformation in roots remains debated ( van Kregten et al ., 2016 ; Nishizawa-Yokoi et al ., 2021 ). These findings highlight that developmental processes may impose different requirements for successful transformation in germline and somatic cells. Understanding the genetic and molecular basis of AMT-FI is crucial to expanding its utility across plant species. Plant immunity plays a pivotal role in suppressing tumorigenesis and AMT efficiencies ( Tiwari et al ., 2022 ; Dodds et al ., 2024 ). Upon Agrobacterium infection, plants recognize pathogen-associated molecular patterns (PAMPs) such as EF-Tu, flagellin, and cold-shock proteins by pattern-recognition receptors (PRRs), activating pattern-triggered immunity (PTI) that suppress transformation ( Zipfel et al ., 2006 ; Wu et al ., 2014 ; Saur et al ., 2016 ; Wang et al ., 2018 ; Fürst et al ., 2020 ). Salicylic acid (SA) is a key defense hormone that reduces tumor formation and transient transformation ( Anand et al ., 2008 ). Additionally, antimicrobial secondary metabolites, including glucosinolates and camalexin, restrict Agrobacterium infection at early and late stages, respectively ( Shih et al ., 2018 ). These defense strategies collectively highlight the resilience of plants to counteract Agrobacterium infection and limit its transformation success. In Arabidopsis , the EF-Tu receptor (EFR) plays a key role in recognizing Agrobacterium EF-Tu and inhibiting transient transformation ( Zipfel et al ., 2006 ; Wu et al ., 2014 ; Wang et al ., 2018 ). However, EFR does not exhibit a significant impact on stable transformation or gene targeting efficiency ( van Tol et al ., 2022 ; Yang et al ., 2023 ). To date, its influence on AMT-FI-mediated genome editing has not been fully elucidated. In this study, we investigated the temporal and spatial localization of Agrobacterium cells and transformation events during AMT-FI in A. thaliana. A dual reporter system, combining a visible RUBY marker with a hygromycin resistance marker, was utilized to identify the floral tissues and stages most susceptible to AMT. Furthermore, we examined the impact of EFR-mediated PTI in transformation and genome-editing efficiencies. Our results revealed that AMT events peak in flowers opened at 6 day- post-inoculation (DPI), with efr mutants exhibiting significantly higher genome editing efficiency than Col-0. Moreover, we engineered two independent Agrobacterium strains expressing a chimeric EF-Tu, which further enhanced both transient expression and genome editing efficiency without significantly altering stable transformation frequency. These findings offer new strategies to facilitate AMT-FI- based genome editing in plants. MATERIALS AND METHODS Plant materials and growth condition Arabidopsis thaliana Col-0, EF-Tu receptor (EFR) mutants, efr -1 (SALK_044334) and efr -2 (SALK_068675) were used in this study. Seeds were sown in soil (peatmoss:vermiculite:perlite = 12:1:1), germinated, and grown in a growth room under conditions of 22 °C with 16h light/8h dark photoperiod. Bacterial growth condition For tracking Agrobacterium localization and transformation events, Agrobacterium cells were cultured in 523 with appropriate antibiotics ( Kado & Heskett, 1970 ). For genome editing experiments, LB medium supplemented with appropriate antibiotics was used. Freshly streaked out Agrobacterium colonies grown on 523 or LB agar were cultured in the same medium (5 mL) for 2 days at 28 °C. The cultures were subsequently diluted 1:1000 in 50 mL 523 or LB, grown for 22-24 hours, harvested by centrifugation at 6000x g for 20 minutes. The cell pellets were resuspended in infiltration medium (1/2 MS, 5% sucrose, 0.04% silwet-L77, 44 nM BAP and 200 μM acetosyringone, pH=5.7) to OD 600 =1. Plasmid construction Plasmid and primer information are listed in Table S1 and S2, respective. 35S::RUBY plasmid (Addgene#160908) which provided a visible RUBY reporter and hygromycin resistance (Hyg R ) reporter was obtained from Addgene. View this table: View inline View popup Download powerpoint Supplemental Table 1. Strain and Plasmid information View this table: View inline View popup Download powerpoint Supplemental Table 2. Primer information The gentamycin resistance gene (Gen R ) of pRL662-GFP(S65T) ( Yu et al ., 2021 ) was replaced by spectinomycin resistance gene from 35S::RUBY to obtain pRL662- GFP(S65T)/Spe R by use of Gibson Assembly® Master Mix (New England Biolabs, USA). The expression of mCherry2 with a 3’-end nuclear localization signal (mCherry-NLS) was generated from pEB2-2ndVal mCherry2-L (Addgene#104027) by PCR. PCR was performed by use of KAPA HiFi HotStart ReadyMix PCR Kit (KapaBiosystems, South Africa) and PCR products were cloned into the pJet1.2/blunt plasmid following the user manual of CloneJET PCR Cloning Kit (ThermoFisher Scientific, USA). 35S::mCherry2-SV40/Kan R binary vector was constructed with Golden Gate Assembly. A3A-CBE coding region was amplified from pYPQ265E2 (Addgene #164719) and was subcloned into XbaI/SacI digested pHEE901 (Addgene #91707) via Gibson Assembly. Protospacers were cloned into pA3A-CBE vectors as previously described ( Steinert et al ., 2015 ). To generate the tufAB Pst4-15 mutants in GV3101, we employed double crossover vector using pEML041003-tufAPst4-15 and pEML41003- tufBPst4-15. Two 2.5-kb DNA fragments of modified tufA and tufB were amplified by two pairs of primers, and cloned into pEML041003 by BsaI digestion. AS201 was generated by modifying tufB followed by tufA , while AS202 was tufA followed by tufB . In both constructs, the amino acids at positions 4-15 of EF-Tu were substituted from the native sequence SKFERNKPHVNI to the Pseudomonas syringae variant EKFDRSLPHCNV. Seedling transient transformation and GUS activity assays A. thaliana seedling transient expression assay was performed by the AGROBEST ( Agrobacterium -mediated enhanced seedling transformation) method as previously described, with minor modification ( Wu et al ., 2014 ). Agrobacterium cultures were adjusted to an OD 600 =0.01 in AB-MES medium for co-culture with seedlings. Infected seedlings were collected for GUS activity assays after 2.5 day-post-inoculation. Agrobacterium -mediated transformation by floral inoculation (AMT-FI) AMT-FI was performed according to the published procedure ( Clough & Bent, 1998 ) with modifications. For tracking Agrobacterium cells and transformation events, each Arabidopsis plant was grown in a small pot (5 cm upper diameter x 5 cm high x 3.5 cm bottom diameter). For genome editing experiments, three plants were grown in a large pot (7.8 cm upper diameter x 7.8 cm high x 5.7 cm bottom diameter). Plants were grown until they reached ∼5 weeks old, with primary inflorescences reaching 10 to 15 cm tall. The primary inflorescence was then removed to promote the emergence of secondary inflorescences over the next 5 to 6 days. Immediately prior to floral dipping, all open flowers were removed. Inflorescences were submerged in Agrobacterium -containing infiltration medium for 40 seconds, then plants were laid horizontally in trays, covered with plastic film to maintain high humidity, kept in the dark and incubated for 20 to 24 hrs. Plants were returned to the growth room under the same growth condition. For time-course experiments, newly opened flowers were labeled daily following inoculation. GFP-labeled Agrobacterium cells and mCherry2 T-DNA reporter signals were examined in ovaries using a Zeiss LSM 510 Meta confocal microscope RUBY signals were monitored visually in plants transformed with GV3101 carrying the 35S::RUBY construct, assisted by a stereo microscope. A Zeiss Lumar V12 stereo fluorescent microscope was used to capture images of floral organs, developing ovules, seeds, and seedlings. To evaluate AMT-FI efficiency by RUBY marker, siliques from RUBY-transformed plants were collected daily. To assess stable transformation efficiency, seeds were sterilized in 70% ethanol for 10 min and plated on a 1/2 MS medium supplemented with 25 mg/L hygromycin. The number of germinated seedlings and Hyg R seedlings was recorded. AMT-FI efficiency was determined based on the proportion of Hyg R seedlings relative to the total number of germinated seedlings. Assessment of genome editing efficiency To simultaneously measure the genome editing and AMT-FI efficiency, seeds from plants transformed with Agrobacterium carrying a binary vector with T-DNA-encoded base editor, a sgRNA and a hygromycin resistance gene were surface sterilized and germinated on a 1/2 MS medium supplemented with either 5 mg/L tribenuron or 25 mg/L hygromycin, both containing 100 mg/L timentin. After seven days, the numbers of tribenuron-resistant, hygromycin-resistant, and albino T1 seedlings were recorded. AMT-FI efficiency was estimated using a weight-based approach, with 10 mg of seeds estimated to contain approximately 500 seeds. Statistical analysis An unpaired two-tailed Student’s t- test, performed using GraphPad Prism 10, was used to assess statistical differences either between Col-0 WT and efr mutants or across time-course datasets. RESULTS Agrobacterium enters the female reproductive organ within one hour but transformation events occur in open flowers after five day-post inoculation Although transformation is thought to occur when the female gametophyte becomes accessible, direct links between floral stage and transformation success remain unclear. To investigate the spatial and temporal dynamics of Agrobacterium infection and transformation, we engineered a fluorescently labeled Agrobacterium strain for live tracking. Agrobacterium GV3101 carrying the disarmed Ti plasmid pMP90 (hereafter referred as GV3101), constitutively expressing GFP marker and a T-DNA expressed mCherry2, was used for floral inoculation in A. thaliana Col-0 (Fig.1a, 1b). GFP-expressing Agrobacterium cells were observed as early as 1 hour-post-inoculation (HPI) in the early developmental stage of open gynoecia, the female reproductive organ lacking central placenta tissue ( Fig. 1c ) but were not detected in the later stage of gynoecia containing central placenta tissue ( Fig. 1d ). This implied that developing placenta may act as a physical barrier to Agrobacterium entry during early infection. By 6 HPI, Agrobacterium cells accumulated in the locule of open gynoecia ( Fig. 1e , 1f), whereas no signal was seen in closed gynoecia ( Fig. 1g ). This indicated that open gynoecia serve as the main entry point of Agrobacterium into the locule before development of female gametophyte. Download figure Open in new tab Figure 1. Agrobacterium enter open gynoecium within 1 hour but transformation events occur in open flowers after 5 day-post inoculation (a) Agrobacterium GV3101 carrying pRL662-GFP(S65T)/Spe R and the binary vector 35S::mCherry2-NLS was used for floral inoculation on Arabidopsis thaliana Col-0 to visualize Agrobacterium localization and transformation events. (b) Schematic illustration of the structure and development of gynoecium from early to mature stage with stigma, style, ovary containing ovules. The floral stages and their corresponding durations were indicated according to Smyth et al. The diagram was created with BioRender.com. After one hour-post-inoculation (HPI), Agrobacterium cells had already entered the locule of developing gynoecium (c) but were absent in later-stage gynoecia containing developed placenta tissue (d). Between 6-9 HPI, more Agrobacterium cells were observed in the locule (e), even at it began to close (f). Agrobacterium was not detected in fully closed gynoecia (g). At one day-post-inoculation (DPI), Agrobacterium cells were detected in young closed ovaries (h) but not in older ovaries undergoing ovule development (i). At 3 DPI (j), Agrobacterium cells persisted in ovaries at late developmental stages. By 5 DPI, Agrobacterium cells were enriched around the micropyle (arrow) (k), though no mCherry2 signal was detected. At floral open stage after 6 DPI, Agrobacterium accumulated around the micropyle, and mCherry2 signal became visible (l and m). Asterisks (*) indicate placenta tissue; arrowheads denote developing ovule; arrows indicate Agrobacterium located around micropyle. Panels (a to k) were merged channel images; panels (I and m) show GFP, mCherry2, DIC and merged views. Scale bar= 100 µm. At one day-post-inoculation (DPI), Agrobacterium cells were detected in locules of younger gynoecia ( Figs. 1h , S1a-f) but were absent in older gynoecia undergoing ovule development ( Figs. 1i , S1g-n). Among more than 30 gynoecia examined, all 13 open gynoecia observed at 6 HPI and 1 DPI showed detectable GFP signals in the locule, indicating the entrance of Agrobacterium prior to the closure of gynoecia. At 3 DPI, Agrobacterium persisted in the locules of more mature gynoecia ( Fig. 1j ), and at 5 DPI, just prior to floral opening (anthesis), Agrobacterium cells were enriched around the micropyles, the ovular opening through which the pollen tube and possibly Agrobacterium gain access ( Fig. 1k ). Despite the sustained presence of Agrobacterium , successful T-DNA expression indicated by mCherry2 fluorescence was not detected before anthesis ( Fig. 1c-k ). Similarly, the RUBY reporter signal was absent in gynoecial tissues until after flower opening ( Fig. 2c ). Following anthesis, mCherry2 signals became visible in cells adjacent to the egg cell/zygote and in the micropyle region where Agrobacterium cells had accumulated ( Fig. 1l , 1m). Transformation signals were also detected in the integument, the ovule’s outer protective layer ( Fig. 1m ). Download figure Open in new tab Figure 2. RUBY marker revealed transformation events in floral organs and female tissues RUBY signals were observed in flowers (a), specifically on the stigma but not on pollen grains (b), and in the transmitting tissue (c). In siliques, RUBY expression was detected in both abortive (d) and developing ovules (e). Flower morphology after AMT-FI ranged from normal open flowers (f) to partially open flowers (g), and flowers with only gynoecia emerging from sepals (h). RUBY signal intensity varied among seeds (i) and plants (j, k). (j) At the seedling stage, hygromycin-resistant (Hyg R ) and RUBY-positive seedling (left), non-transgenic seedling (middle), and RUBY-positive but hygromycin sensitive (Hyg S ) seedling (right) were observed. (k) Transgenic T1 plants with variable RUBY expression patterns from strong whole-plant expression to undetectable signal. Scale bar = 200 µm. Together, these observations reveal that while Agrobacterium rapidly enters the open gynoecium as early as 1 HPI, transformation events are temporally restricted, becoming detectable only in flowers that opened from 5 DPI onward. This correlates with key developmental transitions in ovule accessibility and gametophyte maturity. RUBY-based reporter system uncovers transformation dynamics and floral abnormalities The current method for quantifying transformation efficiency of AMT-FI primarily relies on the use of antibiotic resistance genes, using survival rates of T1 seedlings as a proxy for transformation efficiency. However, this approach overlooks transient transformation events that occur at early stages post-inoculation. To better capture both the transient and stable transformation events, we developed a dual-reporter system combining the visible RUBY marker with a hygromycin-resistance gene ( Fig. 3a ). Download figure Open in new tab Figure 3. Agrobacterium infection and EFR-mediated immunity exert negative impacts on ovule development (a) Experimental design: a schematic diagram illustrates the 35S::RUBY construct and experimental workflow. Transformation efficiency was evaluated based on the presence of RUBY-positive ovules and hygromycin-resistant seedlings. The diagram was created with BioRender.com. (b) Percentage of siliques with RUBY-positive ovules recorded daily for Col-0 WT, efr -1, and efr -2 from flowers that opened at 1-10 DPI. Data are mean ± SD of three biological replicates from two independent experiments. (c) RUBY-based transformation. Percentage of RUBY-positive ovules (include both developed and abortive ovules) per silique collected from flowers of Col-0 WT, efr -1 and efr -2 opened at 5-9 DPI was calculated (all data points shown in Fig. S3a ). Data are mean ± SD of average percentage of RUBY-positive ovules from three independent experiments (circle, triangle and rhombus symbols). Statistically significant differences ( p < 0.05) between Col-0 WT and efr mutants are indicated. (d) Ovule abortion rate. The abortion rate of RUBY-positive, non-RUBY, and total ovules per silique collected from flowers of Col-0 WT, efr -1 and efr -2 opened at 5-9 DPI was calculated (all data points shown in Fig. S3c ). Data are mean ± SD of average abortion rate from three independent experiments (circle, triangle and rhombus symbols). Statistically significant differences ( p < 0.05) between Col-0 WT and efr mutants are indicated. Download figure Open in new tab Figure S1. Basipetal analysis of Agrobacterium distribution along Arabidopsis inflorescence at 1 DPI. A basipetal series of flowers from the inflorescence tip (a) to the first open flower at the base (m and n) were dissected to visualize distribution of Agrobacterium expressing GFP reporter at 1 DPI. The order was determined in considering the position and developmental stage of flowers. Each figure consists of four imaging channels (o). Scale bars indicated 50 µm. Panels a-e show open gynoecia, f-n are closed gynoecia, m and n are from different regions of the same flower. Download figure Open in new tab Figure S2. Number and percentage of Hyg R /RUBY + seedlings. (a) Representative photo of T1 seedlings screening on hygromycin containing 1/2 MS plate. The hygromycin-resistant T1 exhibiting RUBY signals of whole seedling (Hyg R /RUBY + ) was indicated, among the hygromycin-resistant green and larger T1 seedling (Hyg R /RUBY - ), and hygromycin-sensitive green but small seedlings (Hyg S /RUBY - ). The hygromycin-sensitive T1 seedlings with RUBY signals (Hyg S /RUBY + ) shown in Fig. 2j are rarely occurred and not present in this photo. (b) Number and percentage of Hyg R /RUBY + T1 seedlings in Col-0 WT and efr mutants. Total number of Hyg R T1 seedlings (Total) and those exhibiting RUBY signals of whole seedling (RUBY + ) was indicated and the percentage of Hyg R /RUBY + T1s among total Hyg R T1s. Download figure Open in new tab Figure S3. EFR-mediated immunity exerts negative impacts on ovule development and RUBY-based transformation rate GV3101 carrying the 35S::RUBY construct was used for AMT-FI to assess RUBY-based transformation efficiency and ovule abortion rate in Col-0 WT and efr mutants. Siliques from flowers that opened at 5-9 DPI were analyzed to count the total number of ovules, the number of developed ovules with or without RUBY signals, and the number of abortive ovules with or without RUBY signals per silique. (a) Daily RUBY-based transformation rate per silique. The percentage of RUBY-positive ovules (including both developed and abortive ovules) per silique was calculate at each DPI, as well as cumulatively across 5-9 DPI. Each data point represents the percentage of RUBY-positive ovules per silique. (b) Daily RUBY-based transformation rate of developed ovules per silique. The percentage of RUBY-positive developed (non-abortive) ovules per silique was measured at each DPI, as well as cumulatively across 5-9 DPIs. Each data point represents the percentage of RUBY-positive developed ovules per silique. (c) Daily ovule abortion rate per silique. The percentage of abortive ovules (including both RUBY-positive and RUBY-negative) per silique was recorded at each DPI and cumulatively across 5-9 DPI. Each data point represents the percentage of abortive ovules per silique. Data are shown in mean ± SD from three independent experiments, with p -values calculated by Student’s t-test between Col-0 WT and efr mutants. Related to Figure 3 . We first assessed the floral organs exhibiting RUBY expression. The earliest detection of RUBY signals was observed at 4 DPI, localized at the sepals of floral buds ( Fig. 2a ). By 5 DPI, RUBY expression appeared on the stigma of open flowers but not on pollen grains ( Fig. 2b ). Dissection of the ovaries revealed RUBY signal in the transmitting tissues ( Fig. 2c ), as well as in ovules and developing seeds ( Fig. 2d , 2e). While many RUBY ovules were abortive ( Fig. 2d ), others continued to develop normally ( Fig. 2e ). Most flowers developed and opened normally ( Fig. 2f ) while a minor subset exhibited developmental abnormalities. These included flowers with partially open petals ( Fig. 2g ) and those where only the gynoecium emerged from the sepals ( Fig. 2h ). In mature seeds, RUBY expression ranged in intensity from light pink to dark purple ( Fig. 2i ), indicating variable expression levels. In the hygromycin resistance assay, most Hyg R seedlings only showed weak or sparse RUBY signals, with ∼10% or less of T1 seedling showing RUBY in whole seedlings ( Fig. S2 ). Because hygromycin-sensitive seedlings can continue to grow for up to seven days before growth arrest due to their inability to form roots on hygromycin-containing medium, we can still observe RUBY⁺/Hyg S seedlings, although their frequency is low (less than 1% of total transformants, Fig. 2j ). Consequently, not all hygromycin-resistant plants exhibited uniform RUBY expression throughout the plants ( Fig. 2k ). Overall, the percentage of RUBY-positive seeds was higher than the frequency of Hyg R seedlings. This discrepancy may reflect transient expression or expression of RUBY marker in the integument, which does not necessarily indicate stable T1 transformation. Collectively, the dual-reporter system offers a more comprehensive and sensitive tool for monitoring transformation events across the reproductive tissues. The RUBY reporter revealed that AMT-FI enables transformation in a variety of tissues within the female reproductive organ. However, it also uncovered associated developmental abnormalities in flowers and ovules, which in some cases prevent transformed ovules from developing into viable seeds. Agrobacterium infection and EFR-mediated immunity synergistically disrupt ovule development and seed viability Since transformation events were only observed in ovaries after anthesis, we sought to trace the occurrence of AMT events in developing siliques using RUBY reporter as a marker ( Fig. 3a ). Flowers that opened between 1 to 10 DPI were labeled daily. Once embryos were fully developed, approximately 10 days after floral opening and pollination ( Baud et al ., 2002 ), each corresponding silique was examined for RUBY signals in ovules/developing seeds. To assess the impact of EFR-mediated immunity on AMT-FI efficiency, we compared transformation efficiency in Col-0 wild-type (WT) plants and two EFR loss-of-function mutants ( efr -1 and efr -2) ( Zipfel et al., 2006 ). Transformation events were primarily detected in flowers that opened between 5 to 9 DPI, with the highest frequency, nearly 100% of siliques showing RUBY-positive ovules in flowers opened at 6 DPI in both Col-0 WT and efr mutants ( Fig. 3b ). The percentage of siliques harboring RUBY ovules did not differ significantly between WT and efr , suggesting that Agrobacterium can access the open gynoecium and subsequently transform ovules with comparable success across genotypes. However, the proportion of RUBY-positive ovules per silique was moderately higher in efr mutants, suggesting that compromising EFR-mediated PTI can enhance transformation efficiencies ( Figs. 3c, S3a, S3b ). Importantly, the number of siliques per plant and total seed number did not show significant difference between WT and efr mutants ( Fig. S4 ). Thus, the higher RUBY transformation rate per silique in efr mutants ( Figs. 3c , S3b) is not caused by variation of siliques or seeds between Col-0 and efr mutant plants. Download figure Open in new tab Figure S4. Number of siliques, seeds, and hygromycin-resistant seedlings in Col-0 WT, efr -1 and efr -2 mutants after AMT-FI Siliques and seeds developed from flowers that opened at 5-9 DPI in Col-0 WT, efr -1 and efr -2 mutants were analyzed through number of siliques (a) and seeds (b) per plant, and averaged number of hygromycin-resistant (Hyg R ) seedlings per silique (c). Data are mean ± SD of six biological replicates from two independent experiments (circle and triangle symbols). Given that AMT-FI often induced floral abnormalities and ovule abortion, we further investigated whether EFR-mediated PTI affects ovule viability. Strikingly, in Col-0 WT, nearly 60% of RUBY-positive ovules were abortive, indicating a strong negative impact of AMT-FI on ovule development. In efr mutants, although ovule abortion still occurred, the rate was significantly reduced to ∼45%. In contrast, fewer than 20% of non-RUBY ovules were abortive, with no significant difference between Col-0 WT and efr mutants ( Fig. 3d ). On average, Agrobacterium infection resulted in an ovule abortive rate of ∼35% ( Figs. 3d , S3c). Notably, a strong negative correlation was observed between transformation rate and seed viability. Flowers that opened at 6 DPI, despite showing the highest AMT- FI efficiency, produced low number of viable seeds ( Fig. 4a ), likely due to the high abortion rate of RUBY-positive ovules ( Fig. 3d ). However, efr mutants produced significantly more viable seeds per silique than Col-0 WT from flowers that opened at 6 to 8 DPI ( Fig. 4a ), supporting a role for EFR-mediated immunity in transformation- associated ovule abortion. Download figure Open in new tab Figure 4. AMT events occur mainly in flowers opened at 5-9 DPI, peaking at 6 DPI Siliques and seeds developed from flowers that opened at 5-9 DPI in Col-0 WT, efr -1 and efr -2 mutants were analyzed through viability (a) and hygromycin resistance (Hyg R ) screening (b, c, d) and viability (d). Data are (a) average number of viable seeds per silique, (b) percentage of siliques containing Hyg R transformants, (c) percentage of Hyg R transformants and (d) number of Hyg R transformants per plant. Data are mean ± SD of six biological replicates from two independent experiments (circle and triangle symbols). Statistically significant differences ( p < 0.05) between Col-0 WT and efr mutants are indicated. To determine whether the increased number of viable seeds per silique in efr mutants is a consequence of AMT-FI, we quantified seed production in plants treated with infiltration medium, and those inoculated with Agrobacterium GV3101 ( Fig. S5 ). Under both conditions, efr mutants produced more seeds per silique than Col-0 WT ( Fig. S5a , S5b). However, Agrobacterium infection further reduced seed numbers in both genotypes. To assess seed viability, we evaluated germination rates. Seeds from plants treated with infiltration medium germinated at nearly 100% but the germination rate is reduced in both Col-0 and efr (∼90%) with Agrobacterium infection. However, when the number of developed/germinated seeds following Agrobacterium infection is normalized to that of seeds treated with infiltration medium, efr mutants exhibited higher seed development and germination rates ( Fig. S5a ). Download figure Open in new tab Figure S5. Agrobacterium infection and EFR-mediated immunity affect seed outputs. (a) Table showing seed number per silique from plants treated with infiltration medium alone or with Agrobacterium. Percentage of developed/germinated seeds after infection was normalized to those from infiltration medium (IM) treatment. The resulting percentage of developed and germinated seeds indicated the impact on seed viability. (b) Seed counts and germination rates following IM alone or with Agrobacterium IM (IM + GV3101) treatments. Siliques were collected from flowers that opened at 5–9 DPI. The total number of seeds and the number of germinated seeds were compared between treatments. Data were collected from two plants per genotype, except for efr -1 infected with GV3101, where data were obtained from one plant. Data are shown as mean ± SD, with p -values calculated using Student’s t -test to compare Col-0 WT and efr mutants. Taken together, the results suggested that the elevated abortion rates and reduced seed viability observed here are largely attributable to Agrobacterium infection because ovule abortion rates are typically less than 1% under normal growth conditions ( van Tol et al ., 2022 ). EFR-mediated immunity may also reduce seed development and viability but pathogen-induced stress from Agrobacterium infection has a more substantial negative impact than through EFR alone. While alleviation of EFR-mediated immunity reduced the abortion rate among transformed ovules by ∼15%, it did not restore ovule viability to levels observed in non-transformed ovules ( Fig. 3d ). These findings suggest that, in addition to EF-Tu PAMP recognition, other Agrobacterium -derived factors likely contribute to ovule developmental disruption during AMT-FI. Temporal profiling reveals the optimal window for stable AMT-FI transformation Although the RUBY-positive ovules and seeds indicate transformation events, not all develop into viable transgenic T1 seeds. To more accurately evaluate inherited transformation efficiency, we quantified the number of viable transgenic seeds per silique and stable transformation rate using hygromycin resistance as a selection marker. Stable transformation efficiency was calculated based on the number and percentage of Hyg R seedlings collected daily from flowers opened at 5-9 DPI ( Figs. 4 , S4c). As expected, siliques from flowers opened at 6 DPI exhibited the highest transformation efficiency, with an average of ∼90% and a peak at 100% of siliques containing one or multiple Hyg R transformants ( Fig. 4b ). Consistently, stable transformation efficiency peaked at this time point, with an average of ∼26% and a peak at 40% of T1 progeny exhibiting hygromycin resistance ( Fig. 4c ). The number of T1 Hyg R transformants per plant also reached its highest at 6 DPI, averaging ∼55 and peaking at 100 per plant ( Fig. 4d ). These results highlight that flowers opened at 6 DPI as the optimal floral stage for achieving the highest frequency of stable AMT-FI-mediated transformation in Arabidopsis . EFR-mediated immunity suppresses genome editing efficiency Notably, no significant increase in stable transformation efficiency was observed in efr mutants compared to Col-0 WT ( Fig. 4c ), similar to the previous findings ( van Tol et al ., 2022 ; Yang et al., 2023 ). Previous studies have shown that efr mutants exhibit enhanced Agrobacterium- mediated transient expression ( Zipfel et al., 2006 ; Wu, Liu et al., 2014). Since transient expression of CRISPR/Cas9 components is sufficient to induce genome editing without stable T-DNA integration, we next investigated whether EFR-mediated immunity adversely affects gene editing efficiency during AMT-FI. We constructed a T-DNA vector carrying dual selection markers: a hygromycin resistance gene for selecting transgenic plants and a CRISPR/Cas9 system targeting the ALS gene, which confers dominant herbicide resistance ( Chen et al ., 2017 ), for identifying genome-edited plants ( Fig. 5a ). Given that the egg-cell is a major target of AMT-FI ( Desfeux et al ., 2000 ), we adopted a strategy that enables editing in monoploid germline cells at early developmental stages. Specifically, we applied an egg-cell-specific promoter previously shown to induce heritable mutations in the T1 generation ( Wang et al ., 2015 ). To enhance genome editing efficiency and streamline the screening process, we employed an A3A/Y130F-CBE_V01 base editor (termed as A3A-CBE in this study), which has demonstrated efficient C-to-T base editing in both rice and Arabidopsis under a ubiquitin promoter ( Ren et al ., 2021 ). Download figure Open in new tab Figure 5. EFR suppresses genome editing efficiency Agrobacterium GV3101 carrying a binary vector encoding a base editor and a sgRNA was used for floral inoculation in Col-0 WT and efr mutants to assess genome editing and stable transformation efficiencies. (a) A schematic diagram of the binary vector structure for base editing and target gene information. (b) Genome editing efficiency assessed by tribenuron-resistant T1 plants with targeting the ALS Pro197 locus. (c) Stable transformation efficiency evaluated by hygromycin resistance. (b) and (c): Seeds are collected from flowers that opened at 5-9 DPI. Each data point represents biological replicates from six independent experiments. Data are mean ± SD, with p -values calculated by Student’s t-test between Col-0 WT and efr mutants. (d) Genome editing efficiency assessed based on albino phenotype in T1 plants, resulting from by loss-of-function mutations at locus CLA1 and PDS3. Statistically significant differences between Col-0 WT and efr are indicated (* p < 0.05, ** p < 0.01, chi-square analysis). Using this construct, we transformed Col-0 WT and efr mutants and assessed T1 progeny for T-DNA integration (via hygromycin resistance) and genome editing (via tribenuron resistance conferred by ALS editing). The A3A-CBE system achieved an overall editing frequency of approximately 0.5% in total T1 seeds ( Fig. 5b ). Both efr -1 and efr -2 mutants showed higher editing rates, despite the increase being statistically significant only in efr -2. We also evaluated stable transformation efficiency by sowing T1 seeds on a hygromycin-containing medium but no significant increase can be observed in efr mutants ( Fig. 5c ). To further validate these findings, we targeted CLA1 and PDS3 , two genes that cause albino phenotypes when biallelically edited ( Liu et al ., 2022 ). T1 seeds were germinated on a hygromycin-containing medium, and the numbers of green and albino seedlings were quantified ( Fig. 5d ). We observed a slight but consistent increase in the proportion of albino seedlings in efr -1 compared to Col-0 WT, supporting the conclusion that EFR-mediated immunity suppresses genome editing efficiency during AMT-FI. Modification of Agrobacterium EF-Tu enhances transient gene expression and genome editing efficiency but not stable T-DNA integration efficiency To further validate the role of EFR in suppressing genome editing efficiency during AMT-FI, we tested whether an Agrobacterium strain that evades EFR recognition mediates increased genome editing efficiency. To achieve this, we engineered Agrobacterium to express a modified EF-Tu incapable of triggering EFR-mediated PTI. Agrobacterium GV3101 possesses two EF-Tu-encoding genes, tufA and tufB , which produce a translation elongation factor. Our initial attempts to delete both tuf genes were unsuccessful, likely due to their essential function. However, a recent study demonstrated that replacing the DNA fragment encoding entire or N-terminal region (amino acids 1-15) of Agrobacterium EF-Tu with that of Pseudomonas syringae pv. tomato ( Pst ) DC3000 reduced PTI activation and improved transient expression efficiency in A. thaliana leaves ( Yang et al ., 2023 ). Because the first three amino acids of EF-Tu are identical between GV3101 and Pst DC3000, we adopted a similar concept but applied a different experimental strategy to engineer GV3101 by replacing amino acids 4-15 of both tufA and tufB with the corresponding region from Pst EF-Tu, referred to as tufAB Pst4-15 ( Fig. 6a ). We generated two independent tufAB Pst4-15 strains, named as AS201 and AS202, and compared them to the parental GV3101 strain for transient expression, genome editing and stable transformation efficiencies. To assess transient transformation, we transformed A. thaliana seedlings for transient expression analysis using a T-DNA- encoded GUS reporter ( Wu et al ., 2014 ). Both AS201 and AS202 showed significantly higher transient GUS activity in Col-0 WT compared to GV3101 ( Fig. 6b , 6c). As expected, GV3101 exhibited ∼20-fold higher GUS activity in efr -1 than in Col- 0 due to the absence of EF-Tu recognition. However, in efr -1, AS201 and AS202 exhibited lower GUS activity than GV3101, indicating that the engineered EF-Tu proteins may compromise transformation efficiency when PTI is not a barrier for infection. Download figure Open in new tab Figure 6. Modification of Agrobacterium EF-Tu enhances transient gene expression and genome editing efficiency but not stable T-DNA integration efficiency The two engineered Agrobacterium tufAB Pst4-15 strains, AS201 and AS202, were assessed for its efficiency in transient expression, genome editing, and stable transformation in comparison to the parental Agrobacterium GV3101. (a) A schematic diagram illustrating the difference of tufAB sequences at amino acids 4 th to 15 th between Agrobacterium GV3101 and Pseudomonas syringae ( Pst ). (b, c) Comparison of transient transformation efficiencies between the GV3101 WT and two tufAB Pst4-15 strains (AS201 and AS202) in A. thaliana seedlings by quantitative GUS activity (b) GUS staining (c, scale bar =1 cm). Results are representative of three independent experiments. (d) Comparison of genome editing efficiency between the GV3101 WT and two tufAB Pst4-15 strains (AS201 and AS202) measured by the percentage of tribenuron-resistant T1 plants (left) and number of edited T1 plants per three T0 plants (right). (e) Comparison of stable transformation efficiency by the number of hygromycin-resistant T1 plants per three T0 plants, with no significant differences between WT GV3101 and the two tufAB Pst4-15 strains (AS201 and AS202). Seeds were collected from all flowers. Each data point represents biological replicates from three independent experiments. All values are shown as mean ± SD, with p -values calculated by Student’s t-test. Encouraged by these results, we next tested whether the tufAB Pst4-15 strains could improve genome editing efficiencies in Col-0 by AMT-FI. Compared to GV3101, both AS201 and AS202 significantly enhanced genome editing rates and produced more edited T1 plants ( Fig. 6d ). As expected, we observed no significant difference in T- DNA integration rates or in the total number of transgenic T1 plants ( Fig. 6e ). Sequencing-based screening of genome-edited plants Our floral inoculation analysis demonstrated that flowers opened at 6 DPI showed a stable transformation efficiency ∼26% ( Fig. 4c ). Based on this high efficiency, we reasoned that it may be possible to recover genome-edited T1 plants without prior selection for the T-DNA marker ( Fig. 7a ). Download figure Open in new tab Figure 7. Sequencing-based screening of genome-edited plants (a) Workflow for screening genome-edited T1 plants from flowers that opened at 6 DPI. Seeds were germinated in 1/2 MS medium with only supplemented with timentin to suppress Agrobacterium growth without hygromycin selection. 10-day-old T1 plants were analyzed by Sanger sequencing at the ALS or CLA1 loci. The diagram was created with BioRender.com. (b) The number of sequenced T1 plants and editing rates. † indicates the C to T editing at Pro197 locus conferring tribenuron resistance. Statistically significant differences in genome editing (GE) rates between efr -1 and WT Col-0 are indicated (* p < 0.001, chi-square analysis). To test this, we targeted CLA1 gene, which allows easy identification of edited plants based on their albino phenotype, complemented by sequencing analysis. We germinated T1 seeds from Col-0 and efr mutants on MS medium without hygromycin selection. Among these seedlings, we observed biallelically edited albino plants at the frequencies of 3.3% in Col-0, 3.7% in efr -1, and 4.2% in efr -2. We also identified green seedlings carrying monoallelic edits, identified by Sanger sequencing. Their editing rates were 1.6% in Col-0, 15.9% in efr -1, and 4.7% in efr -2 ( Fig. 7b ). To further validate this strategy, we targeted the ALS gene without applying tribenuron selection. Among the T1 seedlings, we identified one edited plant out of 94 T1 seedlings using GV3101 and two out of 93 using the tufAB Pst4-15 mutant, AS201. All three edited T1 plants carried C-to-T base editing at the Pro197 locus, consistent with the expected ALS editing outcome ( Fig. 7b ). In conclusion, genome-edited T1 plants can be obtained without prior selection for T- DNA integrated plants when screening seeds from flowers opened at 6 DPI. Furthermore, evading EFR-mediated PTI enhances genome editing efficiency, further supporting the role of plant immunity in constraining Agrobacterium -based genome engineering. DISCUSSION In this study, we provide mechanistic and practical insights into the Agrobacterium- mediated floral transformation process. By tracking transformation events with a dual-reporter system, we identified the optimal transformation window, leading to the success in obtaining edited plants without prior selection of T-DNA integration. Our findings also reveal EFR-mediated immunity as an important barrier to efficient transient transformation and genome editing via AMT-FI. By integrating floral stage selection, immune evasion, and Agrobacterium engineering, our study provides a practical guideline to advance Agrobacterium- mediated plant transformation and genome engineering. The recommended AMT-FI procedure is summarized in Fig. S6 . First, the engineered tufAB -modified strain AS201, which showed relatively better performance than AS202, is recommended to replace GV3101 for plant transformation in Arabidopsis or other plants possessing EFR . Second, we recommend to collect seeds only from flowers that opened 5∼9 day-post-inoculation (DPI), which yield 4∼5% hygromycin-resistant transformants ( Fig. 4c , 5), compared with ∼2% when seeds are collected from all flowers ( Fig. 6e ). Third, for screening edited T1 plants without prior selection for T-DNA integration, collecting seeds exclusively from flowers opened at 6 DPI can reduce labor and cost as well as increase the frequency of obtaining edited plants ( Fig. 7 ). In our experimental condition, it is critical to follow the experimental procedure for growing only one plant in a small pot (5 cm upper diameter × 5 cm height × 3.5 cm bottom diameter). When growing three plants in one larger pot, the peak of AMT-FI efficiency is generally delayed by approximately one day. Download figure Open in new tab Figure S6. The recommended AMT-FI procedure. The experimental procedures for achieving higher AMT-FI efficiency and for screening genome-edited T1 plants without prior selection for T-DNA integration are illustrated. The engineered tufAB -modified strain AS201 is recommended for plant transformation in A. thaliana or other plant species possessing EFR . The optimized transformation window was determined under the growth conditions of A. thaliana , with each plant cultivated in a small pot (5 cm upper diameter × 5 cm height × 3.5 cm bottom diameter). The diagram was created with BioRender.com. Temporal and spatial tracking of Agrobacterium entry and transformation Using RUBY as a T-DNA reporter, we detected strong RUBY signals in sepal, transmitting tissues, ovules, and developing seeds ( Fig. 2 ). Notably, no RUBY signals were detected in pollen grains, consistent with earlier findings using GUS reporter ( Ye et al ., 1999 ; Bechtold et al ., 2000 ; Desfeux et al ., 2000 ). The variation in transformation efficiency likely reflects cell-type specific susceptibility to Agrobacterium infection or differential transcriptional activity of the CaMV 35S promoter ( Kiselev et al ., 2021 ). Additionally, we observed diverse RUBY expression patterns among hygromycin-resistant transgenic plants. Some plants expressed RUBY throughout their life cycle, while others showed expression only during seedling stages or specific organs. This variability may result from positional effects of T-DNA integration site or differences in promoter activity ( Gelvin, 2003 ; Roca Paixao & Deleris, 2024 ). The presence of hygromycin-sensitive but RUBY-positive seedlings suggests either truncation of the T-DNA at the left border ( Fig. 3a ), where the hygromycin-resistance cassette is located, or the weaker activity of the nos promoter driving expression of the hygromycin-resistance gene. Although various floral tissues and cell types can be transformed ( Figs. 1 , 2), only female gametes serve as heritable targets for producing progenies in Arabidopsis ( Ye et al ., 1999 ; Bechtold et al ., 2000 ; Desfeux et al ., 2000 ). Therefore, Agrobacterium entry into the locule of developing gynoecium is a crucial step in successful transformation ( Desfeux et al ., 2000 ). Our data show that Agrobacterium enters the locule of open gynoecium within hours, colonizes reproductive tissues, and persists around the micropyle of ovules, potentially enabling transformation of egg cells and surrounding tissues. Despite early colonization, transformation events in gynoecium cells only occur in open flowers from 5 DPI onward, coinciding with floral opening and micropyle exposure. Transient T-DNA expression can be observed as early as 1 DPI and highly abundant at 2 DPI when transforming somatic cells like leaves, seedlings, and root explants ( Gelvin, 2003 ; Wu et al ., 2014 ; Lopez-Agudelo et al ., 2024 ). The delayed expression in germ cells may reflect differences in promoter activity, cell-type susceptibility, or route of Agrobacterium entry. Given that pollen tubes access ovules via the micropyle only after floral opening, they may serve as important carriers, facilitating Agrobacterium entry into ovules for transformation. Once inside, Agrobacterium could associate with male gametes or directly attach to egg cells for T-DNA transfer during fertilization. This proposed mechanism explains the high AMT-FI efficiency observed in flowers opening at 5-9 DPI and suggests that transformation occurs in young floral buds 5-9 days before anthesis, supporting previous findings ( Clough & Bent, 1998 ; Ye et al ., 1999 ; Desfeux et al ., 2000 ). AMT peaks in flowers opened at 6 DPI and negatively correlate with seed viability Tracking transformation events by fluorescent marker revealed Agrobacterium targets integument cells ( Fig. 1l , 1m), consistent with previous findings ( Bechtold et al ., 2003 ). However, only the transformed egg cells contribute to transgene inheritance. This explains why RUBY-based transformation rates are significantly higher than those determined by hygromycin resistance ( Figs. 3c , 4c ). The use of the RUBY reporter enabled the discovery of floral stages accessible for AMT-FI, revealing that nearly all ovaries from flowers open at 6 DPI were transformed ( Fig. 3b ), yielding averaged an 26% stable Hyg R T1 progeny ( Fig. 4c ). However, high transformation rates were accompanied by increased ovule abortion, resulting in fewer viable seeds ( Figs. 3d , 4a , 4c ). In contrast, efr mutants exhibited lower abortion rates and higher seed viability per silique, suggesting that EFR-triggered immunity contributes to transformation-induced ovule loss. Evading plant immune responses could therefore enhance the efficiency of transgenic seed production via AMT-FI. Evasion of EFR-mediated immunity enhances genome editing efficiency but not stable transformation Although EFR-mediated immunity was discovered nearly two decades ago ( Zipfel et al ., 2006 ), its influence on stable transformation and genome editing remain unclear until recent studies. While efr -1 mutants showed no improvement on gene targeting or stable transformation efficiencies ( van Tol et al ., 2022 ), they do exhibit enhanced transient expression ( Zipfel et al., 2006 ; Wu, Liu et al., 2014; Yang et al., 2023 ). Our results revealed higher RUBY transformation rates in efr ovules, and confirmed no significant increase in stable transformation measured by hygromycin resistance ( Figs. 4c , 6e ). However, we showed that genome editing efficiencies were higher in efr mutants and in Col-0 WT inoculated with the two engineered GV3101:: tufAB Pst4-15 strain, AS201 and AS202 ( Figs. 5 , 6). This suggests that genome editing can be decoupled from T- DNA integration. This is further supported by evidence that genome-edited plants were successfully obtained from the teb-5 ( polQ) mutant, whereas T-DNA integrated plants were not ( van Tol et al ., 2022 ). Thus, we proposed that enhancement of editing efficiencies may result from higher transient expression of CRISPR/Cas9 in egg cells in the absence of EFR-triggered immunity. Moreover, because editing can occur without DNA integration (e.g., via ribonucleoprotein delivery), it may serve as a proxy for measuring transgene expression levels in germline cells. Innovative strategies for enhancing genome editing efficiency Precision editing is critical for inducing dominant phenotypes by targeting an endogenous gene. However, current methods exhibit low efficiency compared to mutagenesis by Cas9 nuclease ( Huang & Puchta, 2021 ). To overcome these limitations, we used an improved A3A-CBE base editor and achieved a high editing- to-integration ratio (∼0.2) ( Fig. 5 ). This construct, together with our optimized screening workflow, enables efficient dissection factors that influence stable transformation and genome editing efficiency in Arabidopsis . Several recent studies have introduced innovative approaches to enhance stable transformation and genome editing. These include engineering of Agrobacterium strains to deliver type III secretion system effectors, T-DNA concatenation, and vector copy number manipulation ( Raman et al ., 2022 ; Dickinson et al ., 2023 ; Szarzanowicz et al ., 2024 ). Our success in engineering Agrobacterium strains with modified EF-Tu to evade EFR recognition, significantly enhanced genome editing efficiency. This reinforces the idea that boosting transient gene expression is a key determinant for successful genome modification via CRISPR/Cas9. Despite native EFR is specific to Brassicaceae, several non-Brassicaceae crops have been engineered to express EFR for disease resistance ( Lu et al ., 2015 ; Schwessinger et al ., 2015 ; Boschi et al ., 2017 ; Kunwar et al ., 2018 ; Piazza et al ., 2021 ). These transgenic plants may show reduced Agrobacterium- mediated transformation. Our engineered GV3101:: tufAB Pst4-15 strains could overcome this limitation. Beyond EF-Tu, other Agrobacterium- derived PAMPs such as translation initiation factor IF1, cold-shock protein, and flagellin also trigger PTI via distinct receptors in different species ( Saur et al ., 2016 ; Fürst et al ., 2020 ; Fan et al ., 2022 ). This work sets the stage for the development of multi-modified Agrobacterium strains with broad compatibility, offering a roadmap to enhance AMT across diverse plant taxa. AUTHOR CONTRIBUTIONS M.-S.L, T.-K.H., C.-H. K. and E.-M.L, contributed to the conceptualization of the project. M.-S.L. designed and performed AMT-FI. T.-K.H. designed and performed the genome editing assay. M.-S.L., T.-K.H., Y.-C.W. and S.-C.W. performed experiments and analyzed data. M.-S.L., T.-K.H., and E.-M.L. wrote the initial manuscript. E.-M.L., C.-H. K. and C.-H.W. supervised the project. M.-S.L and T.-K.H contributed equally. All authors reviewed and edited the manuscript. SUPPORTING INFORMATION Table S1. Plasmid information Table S2. Primer list Figures S1-S6 DATA AVAILABILITY The data supporting the findings of this study are available within the article or as Supporting Information. Strains and plasmids generated in this study are available either on Addgene (no. 234350-234353, 237864-237866) or upon request ( Table S1 ) CONFLICT OF INTEREST STATEMENT The authors declare no conflict of interest. ACKNOWLEDGEMENTS The authors acknowledge the members of the Lai, Kuo, and Wu labs for discussion and suggestion. We also thank the technical assistance of Live Cell Imaging Core Lab and Sanger DNA sequencing service provided by Genomic Technology Core located at the Institute of Plant and Microbial Biology, Academia Sinica. The funding was supported by the Academia Sinica Grand Challenge Program (AS-GC-111-L02) to E.-M.L., C.-H. K. and C.-H.W, and the intramural fund of the Institute of Plant and Microbial Biology, Academia Sinica. Funder Information Declared Academia Sinica, https://ror.org/05bxb3784 , AS-GC-111-L02 Footnotes msliu{at}gate.sinica.edu.tw ; tengkueih{at}gmail.com ; ejw{at}gate.sinica.edu.tw ; sky972sakura{at}gmail.com ; wuchh{at}gate.sinica.edu.tw ; chk{at}gate.sinica.edu.tw ; emlai{at}gate.sinica.edu.tw In this revision, we have added additional figures (Figures. S4c, S6), in which Figure S6 summarizing a practical guideline and procedure to enhance Agrobacterium-mediated plant transformation and genome engineering. REFERENCES ↵ Anand A , Uppalapati SR , Ryu CM , Allen SN , Kang L , Tang Y , Mysore KS . 2008 . Salicylic acid and systemic acquired resistance play a role in attenuating crown gall disease caused by Agrobacterium tumefaciens . Plant Physiol . 146 ( 2 ): 703 – 715 . OpenUrl Abstract / FREE Full Text ↵ Angulo J , Astin CP , Bauer O , Blash KJ , Bowen NM , Chukwudinma NJ , DiNofrio AS , Faletti DO , Ghulam AM , Gusinde-Duffy CM , et al. 2023 . CRISPR/Cas9 mutagenesis of the Arabidopsis GROWTH-REGULATING FACTOR (GRF) gene family . Front. Genome Ed . 5: 1251557. ↵ Baud S , Boutin J-P , Miquel M , Lepiniec L , Rochat C . 2002 . An integrated overview of seed development in Arabidopsis thaliana ecotype WS . Plant Physiol. Biochem . 40 ( 2 ): 151 – 160 . OpenUrl CrossRef Web of Science ↵ Bechtold N , Jaudeau B , Jolivet S , Maba B , Vezon D , Voisin R , Pelletier G . 2000 . The maternal chromosome set is the target of the T-DNA in the in planta transformation of Arabidopsis thaliana . Genetics 155 ( 4 ): 1875 – 1887 . OpenUrl Abstract / FREE Full Text ↵ Bechtold N , Jolivet S , Voisin R , Pelletier G . 2003 . The endosperm and the embryo of Arabidopsis thaliana are independently transformed through infiltration by Agrobacterium tumefaciens . Transgenic Res . 12 ( 4 ): 509 – 517 . OpenUrl CrossRef PubMed Web of Science ↵ Bélanger JG , Copley TR , Hoyos-Villegas V , Charron JB , O’Donoughue L . 2024 . A comprehensive review of in planta stable transformation strategies . Plant Methods 20 ( 1 ): 79 . OpenUrl CrossRef PubMed ↵ Bevan M . 1984 . Binary Agrobacterium vectors for plant transformation . Nucleic Acids Res . 12 ( 22 ): 8711 – 8721 . OpenUrl CrossRef PubMed Web of Science ↵ Boschi F , Schvartzman C , Murchio S , Ferreira V , Siri MI , Galván GA , Smoker M , Stransfeld L , Zipfel C , Vilaró FL , et al. 2017 . Enhanced bacterial wilt resistance in potato through expression of Arabidopsis EFR and introgression of quantitative resistance from Solanum commersonii . Front. Plant Sci . 8 : 1642 . OpenUrl CrossRef PubMed Chen Y , Wang Z , Ni H , Xu Y , Chen Q , Jiang L . 2017 . CRISPR/Cas9-mediated base-editing system efficiently generates gain-of-function mutations in Arabidopsis . Sci. China Life Sci . 60 ( 5 ): 520 – 523 . OpenUrl CrossRef PubMed ↵ Clough SJ , Bent AF . 1998 . Floral dip: a simplified method for Agrobacterium -mediated transformation of Arabidopsis thaliana . Plant J . 16 ( 6 ): 735 – 743 . OpenUrl CrossRef PubMed Web of Science ↵ Desfeux C , Clough SJ , Bent AF . 2000 . Female reproductive tissues are the primary target of Agrobacterium -mediated transformation by the Arabidopsis floral-dip method . Plant Physiol . 123 ( 3 ): 895 – 904 . OpenUrl Abstract / FREE Full Text ↵ Dickinson L , Yuan W , LeBlanc C , Thomson G , Wang S , Jacob Y . 2023 . Regulation of gene editing using T-DNA concatenation . Nat. Plants 9 ( 9 ): 1398 – 1408 . OpenUrl PubMed ↵ Dodds PN , Chen J , Outram MA . 2024 . Pathogen perception and signaling in plant immunity . Plant Cell 36 ( 5 ): 1465 – 1481 . OpenUrl CrossRef PubMed ↵ Fan L , Fröhlich K , Melzer E , Pruitt RN , Albert I , Zhang L , Joe A , Hua C , Song Y , Albert M , et al. 2022 . Genotyping-by-sequencing-based identification of Arabidopsis pattern recognition receptor RLP32 recognizing proteobacterial translation initiation factor IF1 . Nat. Commun . 13 ( 1 ): 1294 . OpenUrl CrossRef PubMed ↵ Fürst U , Zeng Y , Albert M , Witte AK , Fliegmann J , Felix G . 2020 . Perception of Agrobacterium tumefaciens flagellin by FLS2(XL) confers resistance to crown gall disease . Nat. Plants 6 ( 1 ): 22 – 27 . OpenUrl PubMed ↵ Gelvin SB . 2003 . Agrobacterium -mediated plant transformation: the biology behind the "gene-jockeying" tool . Microbiol. Mol. Biol. Rev . 67 ( 1 ): 16 – 37 , table of contents. OpenUrl Abstract / FREE Full Text ↵ Hooykaas PJJ . 2023 . The Ti plasmid, driver of Agrobacterium pathogenesis . Phytopathology 113 ( 4 ): 594 – 604 . OpenUrl CrossRef PubMed ↵ Huang T-K , Puchta H . 2021 . Novel CRISPR/Cas applications in plants: from prime editing to chromosome engineering . Transgenic Res . 30 ( 4 ): 529 – 549 . OpenUrl CrossRef PubMed ↵ Hwang HH , Yu M , Lai EM . 2017 . Agrobacterium -mediated plant transformation: biology and applications . Arabidopsis Book 15 : e0186 . OpenUrl CrossRef PubMed ↵ Kado CI , Heskett MG . 1970 . Selective media for isolation of Agrobacterium , Corynebacterium , Erwinia , Pseudomonas , and Xanthomonas . Phytopathology 60 ( 6 ): 969 – 976 . OpenUrl CrossRef PubMed Web of Science ↵ Kiselev KV , Aleynova OA , Ogneva ZV , Suprun AR , Dubrovina AS . 2021 . 35S promoter-driven transgenes are variably expressed in different organs of Arabidopsis thaliana and in response to abiotic stress . Mol. Biol. Rep . 48 ( 3 ): 2235 – 2241 . OpenUrl CrossRef PubMed ↵ Kunwar S , Iriarte F , Fan Q , Evaristo da Silva E , Ritchie L , Nguyen NS , Freeman JH , Stall RE , Jones JB , Minsavage GV , et al. 2018 . Transgenic expression of EFR and Bs2 genes for field management of bacterial wilt and bacterial spot of Tomato . Phytopathology 108 ( 12 ): 1402 – 1411 . OpenUrl CrossRef PubMed ↵ Lacroix B , Citovsky V . 2019 . Pathways of DNA transfer to plants from Agrobacterium tumefaciens and related bacterial species . Annu. Rev. Phytopathol . 57 : 231 – 251 . OpenUrl CrossRef PubMed ↵ Liu D , Xuan S , Prichard LE , Donahue LI , Pan C , Nagalakshmi U , Ellison EE , Starker CG , Dinesh-Kumar SP , Qi Y , et al. 2022 . Heritable base-editing in Arabidopsis using RNA viral vectors . Plant Physiol . 189 ( 4 ): 1920 – 1924 . OpenUrl CrossRef PubMed ↵ Lopez-Agudelo JC , Goh F-J , Tchabashvili S , Huang Y-S , Huang C-Y , Lee K-T , Wang Y-C , Wu Y , Chang H-X , Kuo C-H , et al. 2024 . Rhizobium rhizogenes A4-derived strains mediate hyper-efficient transient gene expression in Nicotiana benthamiana and other solanaceous plants . Plant Biotechnol. J . ↵ Lu F , Wang H , Wang S , Jiang W , Shan C , Li B , Yang J , Zhang S , Sun W . 2015 . Enhancement of innate immune system in monocot rice by transferring the dicotyledonous elongation factor Tu receptor EFR . J. Integr. Plant Biol . 57 ( 7 ): 641 – 652 . OpenUrl CrossRef PubMed ↵ Mysore KS , Kumar CT , Gelvin SB . 2000 . Arabidopsis ecotypes and mutants that are recalcitrant to Agrobacterium root transformation are susceptible to germ-line transformation . Plant J . 21 ( 1 ): 9 – 16 . OpenUrl CrossRef PubMed Web of Science ↵ Nam J , Matthysse AG , Gelvin SB . 1997 . Differences in susceptibility of Arabidopsis ecotypes to crown gall disease may result from a deficiency in T-DNA integration . Plant Cell 9 ( 3 ): 317 – 333 . OpenUrl Abstract / FREE Full Text ↵ Nishizawa-Yokoi A , Saika H , Hara N , Lee LY , Toki S , Gelvin SB . 2021 . Agrobacterium T-DNA integration in somatic cells does not require the activity of DNA polymerase θ . New Phytol . 229 ( 5 ): 2859 – 2872 . OpenUrl CrossRef PubMed ↵ Piazza S , Campa M , Pompili V , Costa LD , Salvagnin U , Nekrasov V , Zipfel C , Malnoy M . 2021 . The Arabidopsis pattern recognition receptor EFR enhances fire blight resistance in apple . Hortic. Res . 8 . ↵ Pixley KV , Falck-Zepeda JB , Paarlberg RL , Phillips PWB , Slamet-Loedin IH , Dhugga KS , Campos H , Gutterson N . 2022 . Genome-edited crops for improved food security of smallholder farmers . Nat. Genet . 54 ( 4 ): 364 – 367 . OpenUrl CrossRef PubMed ↵ Raman V , Rojas CM , Vasudevan B , Dunning K , Kolape J , Oh S , Yun J , Yang L , Li G , Pant BD , et al. 2022 . Agrobacterium expressing a type III secretion system delivers Pseudomonas effectors into plant cells to enhance transformation . Nat. Commun . 13 ( 1 ): 2581 . OpenUrl CrossRef PubMed ↵ Ren Q , Sretenovic S , Liu G , Zhong Z , Wang J , Huang L , Tang X , Guo Y , Liu L , Wu Y , et al. 2021 . Improved plant cytosine base editors with high editing activity, purity, and specificity . Plant Biotechnol. J . 19(10): 2052-2068. ↵ Roca Paixao JF , Deleris A . 2024 . Epigenetic control of T-DNA during transgenesis and pathogenesis . Plant Physiol . 197 ( 1 ). OpenUrl CrossRef ↵ Saur IM , Kadota Y , Sklenar J , Holton NJ , Smakowska E , Belkhadir Y , Zipfel C , Rathjen JP . 2016 . NbCSPR underlies age-dependent immune responses to bacterial cold shock protein in Nicotiana benthamiana . Proc. Natl. Acad. Sci. U. S. A . 113 ( 12 ): 3389 – 3394 . OpenUrl Abstract / FREE Full Text ↵ Schwessinger B , Bahar O , Thomas N , Holton N , Nekrasov V , Ruan D , Canlas PE , Daudi A , Petzold CJ , Singan VR , et al. 2015 . Transgenic expression of the dicotyledonous pattern recognition receptor EFR in rice leads to ligand-dependent activation of defense responses . PLoS Pathog . 11 ( 3 ): e1004809 . OpenUrl CrossRef PubMed ↵ Shih PY , Chou SJ , Müller C , Halkier BA , Deeken R , Lai EM . 2018 . Differential roles of glucosinolates and camalexin at different stages of Agrobacterium -mediated transformation . Mol. Plant Pathol . 19 ( 8 ): 1956 – 1970 . OpenUrl CrossRef PubMed Smyth DR , Bowman JL , Meyerowitz EM . 1990 . Early flower development in Arabidopsis . Plant Cell 2 ( 8 ): 755 – 767 . OpenUrl Abstract / FREE Full Text ↵ Steinert J , Schiml S , Fauser F , Puchta H . 2015 . Highly efficient heritable plant genome engineering using Cas9 orthologues from Streptococcus thermophilus and Staphylococcus aureus . Plant J . 84 ( 6 ): 1295 – 1305 . OpenUrl CrossRef PubMed ↵ Schwartz E Suzuki K , Tanaka K , Yamamoto S , Kiyokawa K , Moriguchi K , Yoshida K 2009 . Ti and Ri plasmids . In: Schwartz E ed. Microbial Megaplasmids . Berlin, Heidelberg : Springer Berlin Heidelberg , 133 – 147 . ↵ Szarzanowicz MJ , Waldburger LM , Busche M , Geiselman GM , Kirkpatrick LD , Kehl AJ , Tahmin C , Kuo RC , McCauley J , Pannu H , et al. 2024 . Binary vector copy number engineering improves Agrobacterium -mediated transformation . Nat. Biotechnol . ↵ Tiwari M , Mishra AK , Chakrabarty D . 2022 . Agrobacterium -mediated gene transfer: recent advancements and layered immunity in plants . Planta 256 ( 2 ): 37 . OpenUrl CrossRef PubMed ↵ van Kregten M , de Pater S , Romeijn R , van Schendel R , Hooykaas PJ , Tijsterman M. 2016 . T-DNA integration in plants results from polymerase-θ-mediated DNA repair . Nat. Plants 2 ( 11 ): 16164 . OpenUrl PubMed ↵ van Tol N , van Schendel R , Bos A , van Kregten M , de Pater S , Hooykaas PJJ , Tijsterman M. 2022 . Gene targeting in polymerase theta-deficient Arabidopsis thaliana . Plant J . 109 ( 1 ): 112 – 125 . OpenUrl CrossRef PubMed ↵ Wang Y-C , Yu M , Shih P-Y , Wu H-Y , Lai E-M . 2018 . Stable pH suppresses defense signaling and is the key to enhance Agrobacterium -mediated transient expression in Arabidopsis seedlings . Sci. Rep . 8 ( 1 ): 17071 . OpenUrl CrossRef PubMed ↵ Wang Z-P , Xing H-L , Dong L , Zhang H-Y , Han C-Y , Wang X-C , Chen Q-J . 2015 . Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation . Genome Biol . 16 ( 1 ): 144 . OpenUrl CrossRef PubMed ↵ Weisberg AJ , Wu Y , Chang JH , Lai EM , Kuo CH . 2023 . Virulence and Ecology of Agrobacteria in the Context of Evolutionary Genomics . Annu. Rev. Phytopathol . 61 : 1 – 23 . OpenUrl CrossRef PubMed ↵ Wu H-Y , Liu K-H , Wang Y-C , Wu J-F , Chiu W-L , Chen C-Y , Wu S-H , Sheen J , Lai E-M . 2014 . AGROBEST: an efficient Agrobacterium -mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings . Plant Methods 10 ( 1 ): 19 . OpenUrl CrossRef PubMed ↵ Yang F , Li G , Felix G , Albert M , Guo M . 2023 . Engineered Agrobacterium improves transformation by mitigating plant immunity detection . New Phytol . 237 ( 6 ): 2493 – 2504 . OpenUrl CrossRef PubMed ↵ Ye GN , Stone D , Pang SZ , Creely W , Gonzalez K , Hinchee M . 1999 . Arabidopsis ovule is the target for Agrobacterium in planta vacuum infiltration transformation . Plant J . 19 ( 3 ): 249 – 257 . OpenUrl CrossRef PubMed Web of Science ↵ Yu M , Wang YC , Huang CJ , Ma LS , Lai EM . 2021 . Agrobacterium tumefaciens deploys a versatile antibacterial strategy to increase its competitiveness . J. Bacteriol . 203 ( 3 ): e00490 – 00420 . OpenUrl PubMed ↵ Zipfel C , Kunze G , Chinchilla D , Caniard A , Jones JDG , Boller T , Felix G . 2006 . Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium -mediated transformation . Cell 125 ( 4 ): 749 – 760 . OpenUrl CrossRef PubMed Web of Science References ↵ Chen , Y. , Wang , Z. , Ni , H. , Xu , Y. , Chen , Q. and Jiang , L . ( 2017 ) CRISPR/Cas9-mediated base-editing system efficiently generates gain-of-function mutations in Arabidopsis . Sci. China Life Sci . 60 , 520 – 523 . OpenUrl CrossRef PubMed Koncz , C. and Schell , J . ( 1986 ) The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector . Molec. Gen. Genet . 204 , 383 – 396 . OpenUrl CrossRef Web of Science Liu , D. , Xuan , S. , Prichard , L.E. , Donahue , L.I. , Pan , C. , Nagalakshmi , U. , Ellison , E.E. , Starker , C.G. , Dinesh-Kumar , S.P. , Qi , Y. and Voytas , D.F . ( 2022 ) Heritable base-editing in Arabidopsis using RNA viral vectors . Plant Physiol . 189 , 1920 – 1924 . OpenUrl CrossRef PubMed Quandt , J. and Hynes , M.F . ( 1993 ) Versatile suicide vectors which allow direct selection for gene replacement in Gram- negative bacteria . Gene 127 , 15 – 21 . OpenUrl CrossRef PubMed Web of Science Yu M , Wang YC , Huang CJ , Ma LS , Lai EM . 2021 . Agrobacterium tumefaciens deploys a versatile antibacterial strategy to increase its competitiveness . J Bacteriol 203 : 10 .1128/jb. 00490-20. OpenUrl View the discussion thread. Back to top Previous Next Posted October 12, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. 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