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Mol Plant Pathol
Mol Plant Pathol
10.1111/(ISSN)1364-3703
MPP
Molecular Plant Pathology
1464-6722
1364-3703
John Wiley and Sons Inc. Hoboken

10.1111/mpp.70006
MPP70006
MPP-OA-24-231.R2
Original Article
Original Article
Arabidopsis F‐box proteins D5BF1 and D5BF2 negatively regulate Agrobacterium‐mediated transformation and tumorigenesis
Hu et al.
Hu Qin 1 2
Li Xueying 1 2
Xi Weijie 1 2
Xu Junjie 1 2
Xu Chao 1 2
Ausin Israel 1 2 israel.ausin@gmail.com

Wang Yafei https://orcid.org/0000-0002-3450-7307
1 2 yafeiwang@nwafu.edu.cn

1 State Key Laboratory for Crop Stress Resistance and High‐Efficiency Production College of Life Sciences, Northwest A&F University Yangling Shaanxi China
2 Institute of Future Agriculture Northwest A&F University Yangling Shaanxi China
* Correspondence
Israel Ausin and Yafei Wang, State Key Laboratory for Crop Stress Resistance and High‐Efficiency Production, College of Life Sciences and Institute of Future Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China.
Email: israel.ausin@gmail.com and yafeiwang@nwafu.edu.cn

13 9 2024
9 2024
25 9 10.1111/mpp.v25.9 e7000624 8 2024
14 7 2024
27 8 2024
© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

Abstract

The pathogen Agrobacterium tumefaciens is known for causing crown gall tumours in plants. However, it has also been harnessed as a valuable tool for plant genetic transformation. Apart from the T‐DNA, Agrobacterium also delivers at least five virulence proteins into the host plant cells, which are required for an efficient infection. One of these virulence proteins is VirD5. F‐box proteins, encoded in the host plant genome or the Ti plasmid, and the ubiquitin/26S proteasome system (UPS) also play an important role in facilitating Agrobacterium infection. Our study identified two Arabidopsis F‐box proteins, D5BF1 and D5BF2, that bind VirD5 and facilitate its degradation via the UPS. Additionally, we found that Agrobacterium partially suppresses the expression of D5BF1 and D5BF2. Lastly, stable transformation and tumorigenesis efficiency assays revealed that D5BF1 and D5BF2 negatively regulate the Agrobacterium infection process, showing that the plant F‐box proteins and UPS play a role in defending against Agrobacterium infection.

D5BF1 and D5BF2 suppress the Agrobacterium infection process by mediating the degradation of Agrobacterium virulence protein VirD5 through the host's ubiquitin/26S proteasome system.

Agrobacterium tumefaciens
crown gall
F‐box protein
plant pathogen
virulence protein
National Natural Science Foundation of China 10.13039/501100001809 31801025 32170359 source-schema-version-number2.0
cover-dateSeptember 2024
details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_JATSPMC version:6.4.8 mode:remove_FC converted:13.09.2024
Hu, Q. , Li, X. , Xi, W. , Xu, J. , Xu, C. , Ausin, I. et al. (2024) Arabidopsis F‐box proteins D5BF1 and D5BF2 negatively regulate Agrobacterium‐mediated transformation and tumorigenesis. Molecular Plant Pathology, 25 , e70006. Available from: 10.1111/mpp.70006
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pmc1 INTRODUCTION

Agrobacterium tumefaciens is a soilborne, gram‐negative bacterium from the Rhizobiaceae family. As a pathogen, its infection can cause tumours in a wide range of plant species, the so‐called crown gall disease (Chilton et al., 1977; White & Braun, 1941). It has served as a model organism for studying plant–pathogen interaction and the molecular mechanisms of pathogenesis (Tiwari et al., 2022). A fascinating feature of Agrobacterium is that during the infection process, it delivers a T‐DNA from its tumour‐inducing (Ti) plasmid into host plant cells, and ultimately this T‐DNA integrates into the plant genome (Azizi‐Dargahlou & Pouresmaeil, 2023; Hooykaas, 2023). Different studies showed that the T‐DNA does not select any particular region of the plant genome or chromatin features as its integration sites (Gelvin, 2021; Kim et al., 2007; Shilo et al., 2017). The inserted genes are then expressed in the plant cell (Gelvin, 2017; Nester, 2014).

Additionally, researchers have demonstrated that it is possible to replace the native genes between the left border (LB) and the right border (RB) of the T‐DNA with any DNA of interest. This process can be used to transform plants in laboratory conditions without causing tumours (Caplan et al., 1983; Fraley et al., 1983). This interkingdom gene transfer ability has developed into Agrobacterium‐mediated genetic transformation, an extensively used method in plant biotechnology and crop breeding (Rahman et al., 2023; Thomson et al., 2024).

Agrobacterium can recognize molecules such as acetosyringone (AS) released by the host plant, usually after wounding, and begin the infection with the activation and induction of the virulence proteins encoded by the Ti plasmid (Hooykaas, 2023; Matthysse, 2014; Nester, 2014; Pitzschke & Hirt, 2010). Several studies have demonstrated that during the Agrobacterium infection process, at least five virulence proteins (VirD2, VirD5, VirE2, VirE3 and VirF) are delivered into plant cells together with T‐DNA through the type IV secretion system (T4SS) (Vergunst et al., 2000, 2005). These translocated virulence proteins then interact with plant factors, hijacking host plant systems or pathways to facilitate the cytoplasmic trafficking, nuclear import and the integration of the T‐DNA (Dafny‐Yelin et al., 2008; Djamei et al., 2007; Gelvin, 2010, 2021; Nester, 2014; Tzfira et al., 2001; Wang et al., 2014). Moreover, Agrobacterium requires the host plant's DNA repair machinery, such as polymerase θ‐mediated end joining (TMEJ) and canonical non‐homologous end joining (cNHEJ), to achieve T‐DNA integration into the genomes of infected plants (Kralemann et al., 2022; Thomson et al., 2024).

Agrobacterium infection also causes transcriptional reprogramming in infected plants (Deeken et al., 2006; Ditt et al., 2006; Duan et al., 2018; Lee et al., 2009; Shih et al., 2018). Among the five translocated virulence proteins, VirE3 possesses transcriptional regulation activity in host plant cells (Garcia‐Rodriguez et al., 2006). For example, it suppresses the expression of AtPR1 while activating the expression of AtPDF1.2 and VBF (Li et al., 2021; Niu et al., 2015).

We have previously reported that VirD5 is a putative transcriptional activator‐like effector that binds specifically to the VirD5 response element (D5RE) (Wang et al., 2014). Also, another study showed that an Arabidopsis F‐box protein‐coding gene possessing a D5RE in its promoter is activated by VirD5 (Zhang et al., 2020). VirD5 also binds to Arabidopsis VIP1/2 (VirE2 interacting protein 1/2), two positive regulators of the Agrobacterium infection (Anand et al., 2007; Djamei et al., 2007). Through competitive interaction, VirD5 delays the degradation of VirE2 and VIP1 in plant cells or interferes with their interaction with other proteins (Wang et al., 2014, 2018). Deleting the virD5 gene reduces Agrobacterium‐induced tumour formation and stable transformation efficiency but does not affect the efficiency of transient transformation (Wang et al., 2018). VirD5 is toxic to plant and yeast cells. In yeast, VirD5 binds to the kinetochores by interaction with Spt4, inducing growth inhibition and chromosome mis‐segregation. These phenotypes are caused by the hyperactivation of the mitotic kinase Aurora in host cells (Zhang et al., 2017; Zhang & Hooykaas, 2019). VirD5 can also cause DNA damage and mutations in host cells (Zhang et al., 2022). Thus, the translocated virulence protein VirD5 plays an important role during the Agrobacterium infection process and facilitates stable transformation and tumorigenesis efficiency.

The F‐box E3 ligase and ubiquitin/26S proteasome system (UPS) also play an important role during the Agrobacterium infection process. The Ti plasmid‐encoded protein VirF is an F‐box protein delivered into plant cells and determines the host range (Jarchow et al., 1991; Schrammeijer et al., 1998). VirF interacts with host plant factors, mediating its degradation in plant cells via the UPS (Garcia‐Cano et al., 2015, 2018; Schrammeijer et al., 2001). Interestingly, VirF can also be degraded by the plant UPS, a process that is counteracted by VirD5, which interacts with VirF, preventing the host‐induced degradation of VirF (Magori & Citovsky, 2011).

A previous study demonstrated that the expression of the plant‐specific VIP1‐interacting F‐box protein (VBF) is induced during Agrobacterium infection. VBF functionally replaces VirF in mediating the degradation of VirE2 and VIP1 through a VBF‐containing Skp1‐Cdc53‐F box protein (SCF) complex (Zaltsman et al., 2010). The SCF component proteins ASK1, ASK2, and its accessory protein SGT1 are also induced during Agrobacterium infection and are required for the Agrobacterium‐mediated plant stable transformation (Anand et al., 2012).

The pathogenesis of Agrobacterium has been extensively studied at the pathogen level. Still, there are limited reports about host plant factors and their molecular mechanisms, especially defence mechanisms, during the Agrobacterium infection process. This still needs to be further elucidated. Here, we identified two Arabidopsis F‐box proteins, D5BF1 and D5BF2 (VirD5‐binding F‐box protein 1/2), that interact with VirD5 and mediate its degradation by UPS. Interestingly, the expression of D5BF1 and D5BF2 is repressed by Agrobacterium infection independently of the delivery of the T‐DNA and virulence proteins VirE3 and VirD5. Our findings indicate these two F‐box proteins play a negative regulatory role in Agrobacterium‐mediated stable transformation and tumorigenesis. Consequently, our research showed that host plants employ D5BF1 and D5BF2 as part of their defence strategy against Agrobacterium infection.

2 RESULTS

2.1 VirD5 is degraded by the host plant UPS

To characterize VirD5 in plant cells, we generated Arabidopsis transgenic lines expressing a virD5‐GFP fusion or GFP only driven by a CaMV 35S promoter (35S::virD5‐GFP or 35S::GFP). Although virD5‐GFP transcripts were detected by semiquantitative reverse transcription (RT)‐PCR, the VirD5‐GFP recombinant protein could not be detected in 35S::virD5‐GFP transgenic plants (T1 and T2) as assayed by western blots (Figure S1a,b). However, GFP was detected in the 35S::GFP control lines.

We found that compared to untransformed plants and 35S::GFP transgenic plants, the 35S::virD5‐GFP transgenic plants (T3) were a slightly smaller size during the early development stages (before the sixth leaf). However, they regained the wild‐type size in later development stages (Figure S1c).

A possible explanation for the lack of a detectable VirD5 signal is that it might be degraded due to its toxicity (Zhang et al., 2017). Indeed, the degradation of target proteins by the plant UPS is a common defensive mechanism during the Agrobacterium infection process (Magori & Citovsky, 2011; Wang et al., 2014). Therefore, we speculated that a system for the degradation of VirD5 might exist in the plant cell.

To test this hypothesis, we first used a cell‐free degradation assay. Purified 6 × His‐VirD5 protein was incubated with protein extracts prepared from Nicotiana benthamiana leaves with or without the proteasome inhibitor MG132. The results showed that MG132 significantly promoted the stability of VirD5 (Figure 1a,b), suggesting that the plant UPS degrades VirD5. Consistently, VirD5‐GFP also showed better stability in MG132‐treated leaves of N. benthamiana transiently expressing VirD5‐GFP compared to the untreated control (Figure 1c,d). Together, these data showed that the Agrobacterium virulence protein VirD5 might be targeted for degradation by the host UPS.

FIGURE 1 Degradation of VirD5 by the ubiquitin/26S proteasome system (UPS). (a) Cell‐free degradation assay. The purified 6 × His‐VirD5 protein was incubated with a total extract from Nicotiana benthamiana leaves for the indicated times. The number below the upper picture indicates the relative amount of the target protein. Anti‐actin western blotting (WB) was used to show that the same amount of total extract was used. (b) Quantification of the degradation degree of 6 × His‐VirD5 protein by plant extracts shown in (a). Student's t test was used to analyse the significance of the difference. *p < 0.05; data are expressed as the mean ± SE from three biological replicates. (c) Transient expression of VirD5‐GFP in N. benthamiana leaves by agroinfiltration. GFP was used as a negative control. Anti‐actin WB was used as a loading control. +MG132: with MG132; −MG132: without MG132. (d) Quantification of the WB signals in (c). VirD5‐GFP/GFP signals were normalized to the signals for the internal control actin. Data are expressed as the mean ± SE from four biological replicates. Student's t test was used to estimate the significance between different sets; p‐values are indicated on the graphs.

2.2 Arabidopsis F‐box proteins interact with VirD5

F‐box proteins, including VirF encoded by Agrobacterium and VBF encoded by Arabidopsis, mediate the degradation of target proteins during the Agrobacterium infection process (Tzfira et al., 2004; Zaltsman et al., 2010). It is also known that VirD5 binds to VirF in plant cells (Magori & Citovsky, 2011). Therefore, we aimed to identify putative plant F‐box proteins that could interact with VirD5 to mediate its degradation.

The available transcriptome data shows that the expression of five F‐box protein‐coding genes (At3g58890, At5g42350, At4g02760, At1g31350 and VBF) was altered during Agrobacterium infection (Ditt et al., 2006). Moreover, three F‐box genes (At2g04230, At3g23970 and At3g49480) possessing D5REs in their promoters are known to be regulated by VirD5 (Wang et al., 2014; Zhang et al., 2020). Consequently, we tested the ability of these candidates to interact with VirD5 in planta by bimolecular fluorescence complementation (BIFC) assay. The results showed that VirD5 interacted with two of these F‐box proteins (At1g31350 and At5g42350), here named as VirD5‐binding F‐box protein 1 and D5BF2 (D5BF1 and D5BF2), respectively (Figure 2a).

FIGURE 2 D5BF1 and D5BF2 interact with VirD5 in vitro and in vivo. (a) Bimolecular fluorescence complementation assay to test the interactions between VirD5 and F‐box proteins in Arabidopsis protoplasts. The construct combinations 35S::VirD5‐ceYFP,35S::At2G04230‐neYFP and 35S::Ghd7‐CFP; 35S::VirD5‐ceYFP, 35S::At3G23970‐neYFP and 35S::Ghd7‐CFP; 35S::VirD5‐ceYFP, 35S::At3G49480‐neYFP and 35S::Ghd7‐CFP; 35S::VirD5‐ceYFP, 35S::At1G31350‐neYFP and 35S::Ghd7‐CFP; 35S::VirD5‐ceYFP, 35S::At5G42350‐neYFP and 35S::Ghd7‐CFP were used to co‐transform Arabidopsis protoplasts and the transformed cells were observed with a confocal microscope. YFP, yellow fluorescence protein; CFP, cyan fluorescence protein. ceYFP, C terminal of enhanced YFP. neYFP, N terminal of enhanced YFP. Ghd7‐CFP was used as a nuclear localization marker. BF, bright field. (b) Chitin beads pull‐down assay for testing of D5BF1–VirD5 protein interaction. (c) Glutathione sepharose beads pull‐down assay for testing of D5BF1–VirD5 protein interaction. In (b) and (c), the input and pull‐down products were detected by western blotting (WB) using antibodies as indicated. GST and CBD proteins were used as negative controls. Input, samples processed without pull‐down. GST, glutathione S‐transferase; CBD, chitin‐binding domain; * indicates the degraded VirD5–CBD protein or unspecific protein. (d) Immunoprecipitation assay for testing of D5BF1–VirD5 and D5BF2–VirD5 interaction. The purified 6 × His‐VirD5 proteins were incubated with total proteins extracted from Columbia and transgenic Arabidopsis plants (D5BF1‐FLAG, D5BF2‐FLAG and FAS2‐FLAG), respectively. The FLAG beads were used to precipitate the target protein. The input and immunoprecipitated products (IP) were detected by WB using antibodies as indicated. Columbia and FAS2‐FLAG transgenic plants were used as negative control. Input, samples processed without immunoprecipitation.

To verify the interaction, chitin‐binding domain (CBD)‐tagged VirD5 (VirD5‐CBD) and glutathione S‐transferase (GST)‐tagged D5BF1 (GST‐D5BF1) were heterologously expressed in Escherichia coli BL21 (DE3) cells and in vitro pull‐down assays were performed. The results showed that when the VirD5‐CBD was used as bait, GST‐D5BF1 fusion was precipitated. This was not the case for GST alone. Also, using GST‐D5BF1 as a bait, we could detect the VirD5‐CBD fusion but not CBD. Consistently, when CBD or GST tags were used as bait, the target proteins GST‐D5BF1 or VirD5‐CBD could not be detected. These bidirectional pull‐down assays show that D5BF1 binds VirD5 in vitro (Figure 2b,c).

Unfortunately, we were not able to express D5BF2 in E. coli. However, we could test for immunoprecipitation using Arabidopsis transgenic lines expressing FLAG‐tagged D5BF1 and D5BF2 (Figure S2). Total proteins were extracted from the Columbia (wild type) control and the transgenic plants (D5BF1‐FLAG and D5BF2‐FLAG), then incubated with FLAG magnetic beads and the purified 6 × His‐VirD5 proteins for immunoprecipitation. Western blot assays of the input and immunoprecipitated (IP) samples showed that FLAG‐tagged D5BF1 and D5BF2 coprecipitated with the 6 × His‐VirD5 protein. This seems independent of the FLAG tag because an unrelated FLAG‐tagged protein, FAS2 (FASCIATA2), did not coprecipitate along with VirD5 (Figure 2d). These data demonstrate that plant F‐box proteins D5BF1 and D5BF2 interact with Agrobacterium VirD5 in vivo and in vitro.

2.3 D5BF1 and D5BF2 mediate the degradation of VirD5

To test whether the plant F‐box proteins D5BF1 and D5BF2 act as putative E3 ligases, mediating the degradation of VirD5, a cell‐free degradation assay using purified GST‐D5BF1 and 6 × His‐VirD5 was performed. The results showed that, compared to the control (GST only), the GST‐D5BF1 fusion protein caused a significant decrease in the VirD5 signal (Figure 3a,b).

FIGURE 3 Facilitation of VirD5 degradation by D5BF1 and D5BF2. (a) Cell‐free degradation assay for analysing the stability of 6 × His‐VirD5 protein with purified GST‐D5BF1 or glutathione S‐transferase (GST) protein. The 6 × His‐VirD5 protein was incubated with GST‐D5BF1 or GST protein and total extracts from Nicotiana benthamiana leaves, respectively, for the indicated times. Anti‐actin western blotting (WB) was used to demonstrate that the same amount of total extracts was used in the two sets of assays (+GST‐D5BF1 and + GST). Anti‐GST WB was used to show that the same amount of GST‐D5BF1 or GST protein was used in each tube. (b) Quantification of the degradation degree of 6 × His‐VirD5 protein by N. benthamiana extracts described in (a). Data represent three independent assays. (c) Cell‐free degradation assay using plant extracts from Columbia (wild type), d5bf1–1 and d5bf2–1. Anti‐actin WB was used to show that the same amount of Arabidopsis extracts was used in the three sets of assays. (d) Quantification of the degradation degree of 6 × His‐VirD5 protein by Arabidopsis extracts shown in (c). Student's t test was used to analyse the significance in (b) and (d). *p < 0.05, **p < 0.01; ns, not significant; data are expressed as the mean ± SE from three biological replicates.

We then identified Arabidopsis T‐DNA insertional mutant lines in D5BF1 and D5BF2 (d5bf1‐1, d5bf1‐2, d5bf1‐3 and d5bf2‐1) (Figure S3a,b). None of the available T‐DNA lines for D5BF1 are null mutants. However, the expression of D5BF1 was dramatically reduced in d5bf1‐1 (Figure S3c). In contrast, transcripts of D5BF2 were not detected by reverse transcription‐quantitative PCR (RT‐qPCR) in the d5bf2‐1 background, indicating that it is probably a null mutant (Figure S3d). We used d5bf1‐1 and d5bf2‐1 to repeat the previous cell‐free degradation assay. An equal amount of extracts from the wild type, d5bf1‐1 and d5bf2‐1 were incubated with the same amount of 6 × His‐VirD5 purified protein in this assay. We found that compared to the wild‐type extract, mutant extracts significantly enhanced the VirD5 signal (Figure 3c,d), indicating that D5BF1 and D5BF2 might mediate the degradation of VirD5.

2.4 Agrobacterium infection can repress the expression of D5BF1 and D5BF2 genes

Like other successful pathogens, Agrobacterium is able to modulate the expression of several genes encoded in the host plant genome. To test the effect of Agrobacterium infection on the expression of D5BF1 and D5BF2, we performed agroinfiltration followed by RT‐qPCR. Empty Agrobacterium AGL0 or strains carrying the binary transformation vector (AGL0 vs. AGL0 + pCAMBIA1302; note that pCAMBIA1302 carries Hyg R and GFP) were used to perform agroinfiltration. We observed a decrease in the expression of D5BF1 and D5BF2 48 h after agroinfiltration in AGL0 and AGL0 + pCAMBIA1302, especially in the case of D5BF2 (Figure 4a,b). Nonetheless, the expression level was unaltered at 8 h, 24 h and 32 h after agroinfiltration. We used HIRA (At3g44530) as a negative control, a gene presumably unrelated to the Agrobacterium infection process. The expression of HIRA was not significantly affected by agroinfiltration or the control treatment (infiltration with MES buffer), indicating that Agrobacterium infection represses the expression of D5BF1 and D5BF2 independently of the delivery of T‐DNA (Figure 4a–c).

FIGURE 4 Repression of D5BF1 and D5BF2 genes by Agrobacterium infection. Reverse transcription‐quantitative PCR was used to test the expression of D5BF1 (a and d), D5BF2 (b and e), and HIRA genes (c and f) in Arabidopsis leaves infiltrated with Agrobacterium solution or MES buffer for the indicated times. HIRA and MES buffer were used as negative controls. AGL0 and EHA105 are Agrobacterium strains. AGL0 + pCAMBIA1302, Agrobacterium with a binary vector; EHA105‐ΔvirD5, virD5 gene deletion mutation strain; EHA105‐ΔvirE3, virE3 gene deletion mutation strain. UBQ10 was used as the reference gene. Student's t test was used to estimate the significance between different treatment values. p‐values are indicated on the graphs. ns, not significant.

Besides the T‐DNA, Agrobacterium delivers five virulence proteins into the host plant cells during infection. Two of the five virulence proteins, VirE3 and VirD5, have been proven to have transcriptional regulation activity in plant cells (Niu et al., 2015; Wang et al., 2014; Zhang et al., 2020). Previously, we created Agrobacterium strains lacking virD5 or virE3, EHA105‐ΔvirD5, and ENA105‐ΔvirE3, respectively (Wang et al., 2018). To elucidate the role of VirE3 and VirD5, we examined the expression of D5BF1 and D5BF2 after agroinfiltration with Agrobacterium EH105 and our previously generated mutants. The results showed that D5BF1 and D5BF2 are downregulated upon agroinfiltration in both cases, indicating that the repression of D5BF1 and D5BF2 does not require VirD5 and VirE3 (Figure 4d–f).

2.5 D5BF1 and D5BF2 negatively regulate the Agrobacterium infection process

Agrobacterium has been widely used for the transient and stable transformation of plants. To further examine the function of D5BF1 and D5BF2 during the Agrobacterium infection process, first, we performed a transient transformation assay on d5bf1‐1, d5bf2‐1, and the wild‐type control. The transformation rate was not altered in the mutant backgrounds, indicating that D5BF1 and D5BF2 are dispensable in determining the efficiency of Agrobacterium‐mediated transient transformation (Figure 5a,b).

FIGURE 5 Arabidopsis transient transformation assays. (a) Root segments from each genotype (Columbia, d5bf1‐1, d5bf2‐1 and d5bf1‐1 d5bf2‐1) were inoculated with Agrobacterium EHA105 carrying pBI121 containing uidA as T‐DNA reporter. The inoculated root segments were periodically collected and stained with X‐Gluc. The red bar on the image represents 32 mm. Five replicates were performed. (b) Quantification of the root transformation efficiency described in (a). The transient transformation efficiency was calculated as no. of root segments stained in blue/no. of root segments infected. Error bars in (b) are SEM. ns, not significant.

Our previous work proved that VirD5 is not required for transient transformation but enhances stable transformation (Wang et al., 2018). To test the effect of D5BF1 and D5BF2, we transformed d5bf1 and d5bf2 mutants along with the wild‐type control. Two sets of transformations were performed: one using the vector pCAMBIA1302 carrying Hyg R and a second one using the vector pEG carrying Kan R . Statistical analysis of the transformation efficiency showed that, compared to the wild type (Columbia), all the mutant genotypes tested (d5bf1‐1, d5bf1‐2, d5bf1‐3, d5bf2‐1, d5bf1‐1 d5bf2‐1, d5bf1‐2 d5bf2‐1 and d5bf1‐3 d5bf2‐1) showed significantly higher stable transformation efficiency (Figure 6a). Similar results were obtained using kanamycin as the screening agent in the second transformation set (Figure 6b). An identical assay was performed in parallel using transgenic lines carrying FLAG‐ or GFP‐tagged versions of D5BF1 and D5BF2 under the control of the ACTIN2 promoter (OE‐D5BF1/D5BF2‐GFP/FLAG). In these transgenic lines, stable transformation efficiency was significantly reduced (Figure 6a). Together, these data indicate that D5BF1 and D5BF2 negatively regulate the Agrobacterium‐mediated stable transformation process.

FIGURE 6 Analysis of Agrobacterium infection efficiency. The Agrobacterium‐mediated stable transformation efficiency for each genotype Arabidopsis (wild type: Columbia; single mutants: d5bf1‐1, d5bf1‐2, d5bf1‐3 and d5bf2‐1; double mutants: d5bf1‐1 d5bf2‐1, d5bf1‐2 d5bf2‐1 and d5bf1‐3 d5bf2‐1; overexpression lines: OE‐D5BF1‐FLAG/GFP and OE‐D5BF2‐FLAG/GFP) are shown in (a) and (b). (a) The stable transformation efficiency of each genotype plant transformed by Agrobacterium AGL0 with binary vector pCAMBIA1302. The positive transgenic plants were detected by hygromycin screening. (b) The stable transformation efficiency of each genotype plant transformed by Agrobacterium AGL0 with binary vector pEG. The positive transgenic plants were detected by kanamycin screening. (c) Tumour formation on root fragments infected by wild‐type Agrobacterium A208. Tumours on root segments from each genotype (Columbia, d5bf1‐1, d5bf2‐1, d5bf1‐1 d5bf2‐1, OE‐D5BF1‐FLAG#1, OE‐D5BF1‐FLAG#2, OE‐D5BF2‐FLAG and OE‐D5BF2‐GFP) are shown. OE‐D5BF1‐FLAG#1 and OE‐D5BF1‐FLAG#2 are two independent transgenic lines. (d) Quantification of the stable tumorigenesis efficiency using A208 strain. (e) Stable tumorigenesis efficiency assay using wild‐type Agrobacterium A281 and its virD5 gene deletion mutant A281‐ΔvirD5. The root segments from each genotype with or without tumour formation were counted after 35 days of infection. Student's t test was used to analyse the significance of the difference. *p < 0.05, **p < 0.01; ns, not significant; data are means ± SE from three or five biological replicates.

The Agrobacterium strains used above are engineered strains used in the laboratory for genetic transformation. In nature, Agrobacterium typically infects host plant roots or stems, inducing tumours. To assess the role of D5BF1 and D5BF2 during tumorigenesis, we performed a tumorigenesis efficiency assay on the mutant and overexpressing lines using the wild‐type Agrobacterium strain A208. The results showed that, compared to the wild type (Columbia), the tumorigenesis rate in d5bf1‐1, d5bf2‐1 and d5bf1‐1 d5bf2‐1 backgrounds was significantly increased. In contrast, overexpression of D5BF1 and D5BF2 (OE‐D5BF1/D5BF2‐FLAG/GFP) significantly reduced the tumorigenesis rate (Figure 6c,d), further supporting the hypothesis that D5BF1 and D5BF2 negatively regulate the Agrobacterium infection process.

2.6 D5BF1 and D5BF2 function during the Agrobacterium infection process depends on VirD5

We have demonstrated that D5BF1 and D5BF2 are important factors during the Agrobacterium infection process. To test whether this function depends on VirD5, the tumorigenic strain A281 and a virD5 mutant strain (A281‐ΔvirD5) were used in a tumorigenesis assay on Arabidopsis root segments from the wild type (Columbia), d5bf1‐1, d5bf2‐1, and d5bf1‐1 d5bf2‐1 plants. The statistical analysis of the tumorigenesis rate showed that, in Columbia root segments, the A281‐ΔvirD5 strain rate was significantly reduced compared to the A281 strain (Figure 6e). This is consistent with our previous publication (Wang et al., 2018). The tumorigenesis efficiency was significantly higher in all mutant backgrounds compared to Columbia. This was in agreement with the above result from the A208 strain infection. Interestingly, in the A281‐ΔvirD5 infection assays, all the genotypes (wild type and mutants) showed comparable tumorigenesis efficiency (Figure 6e), indicating that the D5BF1 and D5BF2 function altering the Agrobacterium infection depends on VirD5.

In conclusion, our data prove that the plant‐encoded F‐box proteins D5BF1 and D5BF2, are pivotal in the Agrobacterium infection process, potentially through their role in mediating the degradation of VirD5.

3 DISCUSSION

The ongoing evolving war between pathogens and their host plants involves a series of measures and countermeasures that ultimately determine whether the pathogen or the host wins (Jones & Dangl, 2006). It is becoming evident that the UPS plays an essential role during the pathogen infection and plant defence process. For example, the Agrobacterium F‐box protein, VirF, injected into the host plant during infection, can mediate the degradation of VirE2 and VIP1 using the host UPS (Tzfira et al., 2004). VirE2 and VIP1 proteins are important for the protection and trafficking of the T‐DNA into the plant nucleus (Djamei et al., 2007; Gelvin, 1998; Gusev et al., 2020; Tzfira et al., 2001). Still, they may also represent a barrier to the chromosomal integration of the T‐DNA. So, their degradation by the UPS is critical for integrating the T‐DNA. Fascinatingly, there is a plant‐encoded F‐box protein, VBF, that can functionally replace VirF in degrading VirE2 and VIP1 through the UPS and facilitating the infection process (Zaltsman et al., 2010).

Moreover, some other plant‐specific F‐box proteins are also involved in the targeted proteolysis of the T‐complex (Anand et al., 2012). These studies suggested that Agrobacterium uses F‐box proteins to hijack the host UPS and promote infection. In this study, we have found two F‐box proteins, D5BF1 and D5BF2, encoded by Arabidopsis, that probably mediate the degradation of VirD5 through the UPS, thus counteracting Agrobacterium infection. This indicates that the plant UPS is not only abused by Agrobacterium but can also function as a defence against Agrobacterium infection. So, it could be one of the contending focuses in the Agrobacterium–plant war.

Even though D5BF1 and D5BF2 both have an F‐box domain as part of the F‐box superfamily, they are not very similar (only 21.38% identity). However, phylogenetic tree analysis indicates that D5BF1 and D5BF2 are evolutionarily conserved, as they are found in algae, monocotyledonous, and dicotyledonous plants (Figure S4). It is well‐known that Agrobacterium can transform monocotyledonous and dicotyledonous plants in laboratory conditions. However, the transformation frequency between these plants is significantly different (Azizi‐Dargahlou & Pouresmaeil, 2023). Further investigation is still needed to discern whether D5BF1 and D5BF2 or their homologues correlate with the susceptibility to Agrobacterium in different plant lineages.

D5BF1 is also known as KFU1 (Karrikin Upregulated F‐box 1), whose expression can be activated by strigolactone and karrikin. KFU1 is involved in the regulation of hypocotyl elongation and drought tolerance (Sepulveda et al., 2022; Tian et al., 2022; Wang et al., 2020). To date, there is no evidence to show that strigolactone and karrikin signalling pathways are associated with the Agrobacterium infection process. It will be interesting to explore the possible connection between them in the future. D5BF2, also known as CFK1 (COP9 Interacting F‐box Kelch 1), regulates hypocotyl elongation and DNA methylation through the degradation of its target via UPS (Chen et al., 2021; Franciosini et al., 2013). The combination of this data with the present study points to the role of D5BF1/KFU1 and D5BF2/CFK1 in the degradation of target proteins by the UPS and its vital function in plant development and defence against pathogen infection.

When using hygromycin to quantify the stable transformation efficiency of double mutants (d5bf1‐1 d5bf2‐1, d5bf1‐2 d5bf2‐1, and d5bf1‐3 d5bf2‐1), the efficiency was significantly higher than in the single mutant d5bf2‐1, but similar to the corresponding single mutants d5bf1‐1, d5bf1‐2 or d5bf1‐3. However, we did not detect any significant difference between double and single mutants in the stable transformation efficiency assay using kanamycin and tumorigenesis assays. Nonetheless, their efficiency is significantly higher in all cases than in wild‐type plants. These data indicated that D5BF1 and D5BF2 could play independent roles in the defence against Agrobacterium infection.

Despite the significant reduction in infection efficiency caused by the overexpression of D5BF1 and D5BF2, it did not completely prevent the Agrobacterium infection process. This aligns with prior research indicating that VirD5 is important for typical infection efficiency, but not crucial for the Agrobacterium infection process itself (Wang et al., 2018) and supports that the function of D5BF1 and D5BF2 during the Agrobacterium infection depends on VirD5.

Our data showed that D5BF1 and D5BF2 might have evolved to mediate the degradation of VirD5, which can facilitate the Agrobacterium infection process. However, Agrobacterium has also evolved a pathway to repress D5BF1 and D5BF2 expression during infection. In this study, the D5BF1 expression pattern was consistent with a previous publication (Ditt et al., 2006), while other studies reporting transcriptomic data did not detect significant changes in D5BF1 upon Agrobacterium infection (Lee et al., 2009; Shih et al., 2018). Two previous studies also report that D5BF2 is upregulated upon Agrobacterium infection (Ditt et al., 2006; Lee et al., 2009), while another study did not detect a significant change in the expression of D5BF2 (Shih et al., 2018). Different plant materials, methods, and strains might explain these apparent discrepancies in the case of D5BF2. It is worth noting that whole genome transcriptomic data might not be as accurate in detecting moderate changes as testing individual loci by RT‐qPCR. With that in mind, we have used living plants, rigorous controls and two different strains to reinforce the reliability of our data. Moreover, our data indicate that the observed suppression is not the result of the T‐DNA being delivered into host plant cells but the Agrobacterium attack itself.

Two virulence proteins, VirE3 and VirD5, have transcriptional regulatory activity in the host plant cells. However, our data indicates that these proteins are not necessary in repressing D5BF1 and D5BF2. We suspect that Agrobacterium may exploit a plant factor or system currently unknown to suppress the expression of D5BF1 and D5BF2 during infection, as it relies on host factors or systems to facilitate its infection.

We tried to detect VirD5 in Arabidopsis transgenic plants. Even in the lines expressing VirD5 under the constitutive promoter CaMV 35S, we could not detect VirD5 at the protein level. This suggests that VirD5 may be degraded by the host plant, possibly due to its toxicity for eukaryotic cells (Zhang et al., 2017). Therefore, we hypothesize that the host plant cannot tolerate high concentrations of VirD5 in the cells, and thus, it is targeted for degradation, probably through the UPS.

We previously found that VirD5 enhances Agrobacterium infection (Wang et al., 2018). Therefore, an unknown pathway must have evolved to suppress the expression of the key E3 ligases D5BF1 and D5BF2 to attenuate VirD5 degradation by the UPS. In this context, VirD5 binds VirF, hindering its degradation by the host plant UPS (Magori & Citovsky, 2011). Prior VirD5 degradation by the host cell is probably required for the efficient degradation of VirF.

In brief, our research shows that the host plant UPS, including F‐box proteins D5BF1 and D5BF2, plays an important role during the Agrobacterium infection and the subsequent plant defence processes. Both the host plant and Agrobacterium have evolved F‐box proteins in an attempt to gain an advantage in their ongoing battle.

4 EXPERIMENTAL PROCEDURES

4.1 Plant materials and Agrobacterium strains

Arabidopsis plants (Col‐0 ecotype) and N. benthamiana plants were grown at 22°C under long‐day conditions (16 h light, 8 h dark). The following Arabidopsis mutants were used: d5bf1‐1 (SALK_206003C), d5bf1‐2 (CS821340/SAIL_506_G09), d5bf1‐3 (CS811065/SAIL_238_F12), d5bf2‐1 (CS875345/SAIL_613_C07). All the mutant lines were ordered from ABRC. The Agrobacterium strains EHA105 (Urban et al., 1994), AGL0 (Schaart et al., 1995), A208 (Wirawan et al., 1993) and A281 (Hood, Chilton, et al., 1986; Hood, Helmer, et al., 1986) are stored in our laboratory. virD5 gene deletion mutation strains EHA105‐ΔvirD5 and A281‐ΔvirD5 were described in our previous publication (Wang et al., 2018). virE3 gene deletion mutation strain EHA105‐ΔvirE3 was generated as described in our previous publication (Wang et al., 2018).

The full‐length cDNAs of virD5, D5BF1 and D5BF2 were cloned into the binary vector pCAMBIA1302 (Hajdukiewicz et al., 1994; Höfig et al., 2003) and modified pCAMBIA3301 (Liu et al., 2017) for the construction of Arabidopsis transgenic lines (35S::virD5‐GFP and P ACTIN2 ::D5BF1/D5BF2‐FLAG/GFP) by the floral dip method. The empty pCAMBIA1302 was used to transform Arabidopsis to obtain 35S::GFP transgenic lines.

All the agrobacterial strains and plasmids used in this study are listed in Table S1.

4.2 Cell‐free degradation assay

The cell‐free degradation assay was conducted following the methods described in previous studies (Magori & Citovsky, 2011; Wang et al., 2014). Each assay was repeated at least three times to ensure reliability. Quantification of the immunoblot signals was achieved using GelScan software. Statistical analysis and graph generation were performed using GraphPad Prism v. 9.0.

4.3 Transient expression by agroinfiltration in tobacco leaves

The Agrobacterium strain EHA105 carrying the binary vector (35S::virD5‐GFP or 35S::GFP) was grown in Luria Bertani (LB) medium with antibiotics at 28°C. Agrobacterium cells were harvested by low‐speed centrifugation (1000 g) at room temperature and resuspended to OD600 of 0.8 in infiltration buffer (10 mM MgCl2, 10 mM MES, 100 mM acetosyringone). The bacterial suspension was incubated for 2 h at room temperature. Subsequently, it was infiltrated into the abaxial sides of approximately 2‐week‐old N. benthamiana leaves using a 1‐mL needleless syringe. After 48 h growth, the same leaves were treated with 100 μM MG132 or an equivalent volume of dimethyl sulphoxide (DMSO), both diluted in an infiltration buffer lacking acetosyringone. Following an additional 12 h of growth, the infiltrated leaves were collected for protein extraction and subsequent western blot analysis. A GFP‐specific antibody was used for detection. As an internal control, a western blot with an anti‐actin antibody was performed.

4.4 Bimolecular fluorescence complementation

For the BIFC assay, the full‐length coding sequences of VirD5 and Arabidopsis F‐box proteins were cloned in‐frame into the pSPYVE(M) or pSPYNE173 vectors (Waadt et al., 2008), respectively. Our previous publication described the protoplast transformation and fluorescence signal detection procedure (Wang et al., 2014, 2018).

4.5 In‐vitro pull‐down and immunoprecipitation

For in‐vitro pull‐down assay, the full‐length coding sequence of VirD5 and Arabidopsis F‐box proteins were cloned in‐frame into pTXB3 (New England Biolabs) and pGEX‐6p‐1 (Cytiva), respectively, to express the CBD and GST fusion protein in E. coli BL21 (DE3). The pull‐down methods were described in the previous publications (Wang et al., 2014, 2018). For the immunoprecipitation assay, 2 g of each genotype Arabidopsis plants (Columbia, D5BF1‐FLAG, D5BF2‐FLAG and FAS2‐FLAG) were used for extraction of the total proteins in 10 mL IP buffer (50 mM Tris pH 7.6, 150 mM NaCl, 5 mM MgCl2, 10% glycerol, 0.1% NP‐40, 0.5 mM dithiothreitol [DTT], 1 mM PMSF, 1× protease inhibitor), respectively. Total proteins were incubated with the same amount (70 μL) of FLAG magnetic beads (Sigma) at 4°C with slow rotation for 2 h. The beads were washed with IP buffer once for 5 min with rotating, then resuspended in 1 mL IP buffer. Twenty microlitres of purified 6 × His‐VirD5 protein was incubated with the resuspended beads at 4°C under gentle rotation for 1 h. After six times washing with IP buffer, the beads were resuspended with 30 μL phosphate‐buffered saline (PBS) and 30 μL of 2 × SDS sample buffer (4% SDS, 0.2% bromophenol blue, 100 mM Tris‐Cl pH 6.8, 20% glycerol, 200 mM DTT). The precipitated proteins were released by boiling the beads for 6 min. The released proteins were analysed by western blotting using anti‐His (1:3000) and anti‐FLAG (diluted 1:5000) antibodies. The wild‐type and unrelated FAS2‐FLAG transgenic line were used as a negative control in this experiment.

4.6 Agroinfiltration of Arabidopsis leaves and gene expression testing

The Agrobacterium strains (AGL0, EHA105, EHA105‐ΔvirD5 or EHA105‐ΔvirE3) with or without a binary vector pCAMBIA1302 were grown in LB medium at 28°C. Agrobacterium cells were harvested using low‐speed centrifugation at 1000 g for 10 min at room temperature. The pellet was then resuspended to an OD600 of 0.2 in MES buffer (10 mM MES, 100 mM acetosyringone). The bacterial suspension was incubated for 2 h at room temperature. Following incubation, it was infiltrated into the abaxial sides of 3‐week‐old Arabidopsis thaliana (Columbia) leaves using a 1‐mL needleless syringe. At 8, 24, 32 and 48 h post‐infiltration, the leaves were harvested for total RNA extraction. The extraction was performed using the RNA Prep Pure Plant Kit (Tiangen) according to the manufacturer's instructions. We used approximately 2 μg of RNA to synthesize cDNA using the PrimeScript Reagent Kit with gDNA Eraser (TaKaRa). The sample without agroinfiltration, designated as 0 h, was used as a control. We performed signal detection, quantification and normalization using Quant Studio 6 proprietary Software (Life Technologies). In this experiment, the MES buffer and the unrelated HIRA gene were employed as negative controls for the treatment. For all quantitative analyses, we conducted three to four biological replicates. Statistical analysis and graph generation were performed using GraphPad Prism v. 9.0 software.

4.7 Agrobacterium‐mediated Arabidopsis root transient transformation assay

Arabidopsis root transient transformation and β‐glucuronidase (GUS) histochemical staining experiments were performed basically as previously described by Li et al. (2005). The EHA105 strain carrying the binary vector pBI121 containing uidA gene within the T‐DNA region (Chen et al., 2003) was used to infect Arabidopsis root segments. Before infection, the bacteria were resuspended in the transformation suspension medium (Wang et al., 2015) and adjusted to OD600 = 0.1. About 60 root segments from each genotype (Columbia, d5bf1‐1, d5bf2‐1 and d5bf1‐1 d5bf2‐1) were infected. The transient transformation efficiency was calculated as number of root segments stained in blue/number of root segments infected. Five biological repeat data were used for statistical analysis.

4.8 Agrobacterium‐mediated Arabidopsis stable transformation efficiency assay

The transformation and analysis of transformation efficiency were conducted following the methods described in previous publications (Bilichak et al., 2014; Mestiri et al., 2014). Agrobacterium strain AGL0 with binary vector pCAMBIA1302 (with Hyg R ) or pEG (with Kan R ) (Earley et al., 2006) were used for the stable transformation of Arabidopsis by floral dip. The bacteria solution was adjusted OD600 to about 1.0 for transformation. Eighteen plants of each genotype were used for stable transformation, and about 500 mg transgenic seeds (T1) were used for transformation efficiency assay in each biological repeat. Hygromycin (40 μM) and kanamycin (50 μM) were used to screen positive transgenic plants. Three biological repeats were done for statistical analysis. The statistical analysis and graphs were done using GraphPad Prism v. 9.0 software.

4.9 Tumorigenesis assay

The Agrobacterium strains A208, A281 and A281‐ΔvirD5 were used for tumorigenesis assays. The tumorigenesis assay was performed as described in previous publications (Tenea et al., 2009; Wang et al., 2018). More than 50 root segments from about 20 plants of each genotype were used for infection. Before infection, the bacteria were resuspended in the transformation suspension medium (Wang et al., 2015) and adjusted to OD600 = 0.2. Three to five biological repeats were done for statistical analysis. Statistical analysis and graph generation were performed using GraphPad Prism v. 9.0 software.

Supporting information

Figure S1. VirD5 protein cannot be detected in transgenic Arabidopsis plants. 35S::virD5‐GFP and 35S::GFP were used to transform Arabidopsis (Columbia) plants, respectively. The expression level of target transgene in T1 (a) and T2 (b) was tested by semiquantitative reverse transcription‐PCR and western blot (WB). (c) The phenotype of transgenic plants growing under long days (16 h light, 8 h dark) at 22°C.

Figure S2. Construction of the overexpression transgenic lines. (a) Reverse transcription‐quantitative PCR was used to detect the expression level of the target gene (D5BF1 or D5BF2) in transgenic lines used in this research. (b) Anti‐FLAG western FLAG and OE‐D5BF2‐FLAG. (c) Anti‐GFP WB was used to detect the transgenic lines OE‐D5BF1‐GFP and OE‐D5BF2‐GFP.

Figure S3. Identification of Arabidopsis T‐DNA insertion mutants for D5BF1 and D5BF2 genes. (a) Genes’ structure diagrams of the D5BF1 and D5BF2 loci. The T‐DNA insertion sites for each mutant allele and primers for genotyping are denoted. (b) Genotyping of the mutant lines. (c) Testing of D5BF1 gene expression level in each mutant by reverse transcription‐quantitative PCR (RT‐qPCR). (d) Testing of D5BF2 gene expression level in the mutant by RT‐qPCR.

Figure S4. Evolutionary conservation analysis of D5BF1 and D5BF2 in plants. (a) Phylogenetic tree of D5BF1 and its orthologues from representative monocotyledonous, dicotyledonous plants and algae. (b) Phylogenetic tree of D5BF2 and its orthologues from representative monocotyledonous, dicotyledonous plants and algae. The evolutionary history was inferred using the neighbour‐joining method. The optimal tree is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. All ambiguous positions were removed for each sequence pair (pairwise deletion option). Evolutionary analyses were conducted in MEGA 11. This analysis involved 32 (a) and 23 (b) amino acid sequences. The final dataset had 1435 (a) and 1150 (b) positions.

Table S1: Agrobacterial strains and plasmids were used in this study.

ACKNOWLEDGEMENTS

We thank Fei Huang and Shaojun Zhang in Professor Meizhong Luo's laboratory for their help in constructing the EHA105‐ΔvirE3 mutant strain and for their helpful discussions. This work was supported by the National Science Foundation of China (grant numbers 31801025 and 32170359).

DATA AVAILABILITY STATEMENT

The original contributions presented in the study are included in the manuscript, and in its Supporting Information, further inquiries can be directed to the corresponding authors.
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