1. Introduction
[Research background] Plant disease resistance response is a complex process in which a series of genes, signaling pathways, and multi gene products are activated. Generally speaking, disease resistant genes activate downstream defense responses after identifying Avr genes. Studying the disease resistance response pathway activated by the Arabidopsis disease resistance gene L5 (AT1G12290) based on yeast two-hybrid technology is beneficial for elucidating the resistance mechanism of the disease resistance gene, revealing the mechanism of disease occurrence, and laying a theoretical foundation for integrating this gene to cultivate long-lasting and broad-spectrum disease resistant new rice varieties in the future.
Plants have evolved two levels of receptor-based immune systems to battle with multifarious pathogens [1]. Plants employ membrane-localized pattern recognition receptors to monitor conserved microbe-associated molecular patterns (MAMPs), such as bacterial flagellin and fungal chitin, known as MAMP triggered immunity (PTI), which can impede the growth of nonpathogens [2]. Some pathogens suppress PTI by delivering virulence effectors to plant cells, making plants susceptible to diseases. To counter the functions of effectors, plants deploy intracellular nucleotide-binding sites and leucine rich repeat (NBS-LRR) proteins launch the second layer of immunity through recognize effectors and is termed effector-triggered immunity (ETI), resulting in localized hypersensitive-response (HR) cell death [3].
NBS-LRR is mainly divided into two types: CC-NBS-LRR (CNL) and TIR-NBS-LR (TNL) [4]. The function of the NBS-LRR domain has been extensively studied, but the mechanism of NBS-LRR activation and subsequent signal transduction is not yet clear. Some NBS-LRR CC or TIR domains, such as ZAR1, MLA10, L3, L5, SUT1 and L6, are enough to induce cell death when they are transiently expressed in N. benthamiana [5]-[10]. The pre-activated NBS-LRR protein has been localized in multiple subcellular compartments. The activation of NBS-LRR is related to dynamic repositioning. In some cases, a small portion of the total NBS-LRR library appears to be repositioned to the nucleus, where it is believed to regulate defense gene transcription [11] [12]. However, the mechanisms regulating these NBS-LRR are far from understood.
[Research theme] L5 is the CC-NBS-LRR immune receptor [8] [13] [14]. The use of yeast two-hybrid (Y2H) system to study the network of protein-protein interactions is a commonly used strategy [15] [16], which also plays an important role in the study of plant pathogen interaction [17]. Using yeast two hybrid technology to identify proteins in Arabidopsis that directly interact with L5, revealing the disease resistance signaling pathway directly involved by L5, providing a theoretical basis for the next step of multi disease resistance gene aggregation to cultivate broad-spectrum, persistent, and high resistance rice varieties.
2. Materials and Methods
2.1. Experimental Materials
The Arabidopsis and Nicotiana benthamiana were grown in a greenhouse at the Fuzhou Medical University. Briefly, Arabidopsis and N. benthamiana were sown in sterile vermiculite containing diluted Hoagland solution under a 16 hours light period. Yeast strains Y2HGold and Y187, as well as plasmid vectors pDEST-GADT7 (pGADT7, AD) and pDEST-GBKT7 (pGBKT7, BD), were obtained as gifts from Professor Guangcun He (College of Life Sciences, Wuhan University).
2.2. mRNA Extraction and Reverse Transcription
Total RNA was acquired from 4-week-old wild-type Arabidopsis leaves using Trizol reagent and chloroform. Precipitate RNA with isopropanol and dissolve it in RNase-free water. Remove DNA contamination by treating RNA solution with RNase-free DNase at 37˚C for 30 minutes. Use the Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific) to synthesize first stranded cDNA from total RNA at 42˚C for 60 minutes. Amplify target CDS from cDNA for cloning and transient expression.
2.3. Plasmid Construction
Simply put, use the cDNA of wild-type Arabidopsis leaves as the initial template. Transfer the recovered PCR products into the entry vector PENTR/D through a one-step cloning method. Subsequently, the target fragment was transferred to the expression vector using Gateway cloning technology, such as yeast vector pGBDKT7 and pGADT7 or plant vector pEarleygate101 for YFP-HA or Myc tagged.
2.4. Yeast Two-Hybrid Assay
Linking the CC domain of L5 with the bait vector pGBKT7 for screening 4-week-old wild-type Arabidopsis leaves cDNA library constructed in the prey plasmid pGADT7. The transformation, mating, screening, and interaction assays of yeast were strictly carried out in accordance with the corresponding chapters in the Clontech Yeast Protocols Handbook.
2.5. Agroinfiltration in Nicotiana Benthamiana
In a word, A. tumefaciens GV3101 carrying the construct was incubated overnight in liquid LB medium containing kanamycin and rifampicin, and suspended in MMS solution [10 mmol MgCl2, 10 mmol MES (pH = 5.6)]. The Agrobacterium suspension was permeated into the leaves of N. benthamiana.
2.6. Subcellular Localization
In a nutshell, a laser confocal fluorescence microscope (Leica SP8) was exploited to observe photographs of live cells on the abaxial sides of N. benthamiana leaves at 48 h post infiltration (hpi).
2.7. Trypan Blue Staining
N. benthamiana leaves were boiled for 5 min in a 1:1 mixture of absolute alcohol and staining solution (100 ml lactic acid, 100 ml phenol, 100 ml glycerol, 100 ml H2O and 100 mg Trypan blue). Leaves were destained in 2.5 g∙ml−1 chloral hydrate in water.
2.8. Gene Accession Number
The sequence information of the genes used in this article can be identified under GenBank accession numbers AT1G12290 (L5), AT1G01050 (PPA1), AT3G25070 (RIN4), AT3G49580 (LSU1), AT3G51960 (BZIP24), BOI (AT4G19700), RING/U (AT4G22250) and PPA3 (AT2G46860).
3. Results
3.1. Screening of Interaction Proteins of AtL5
The full-length L5 protein (884 amino acids) and its CC domain (160 amino acids) can both cause macroscopic cell death in N. benthamiana [8]. Considering that full-length L5 is not conducive to yeast two hybrid screening, we chose its N-terminus as a substitute. In order to avoid false positive clones, we first conducted experiments on whether BD-L5 CC has self-activation activity. We co-transformed two yeast plasmids, BD-L5 CC and AD-T, as well as BD-P53 and AD-T as positive controls, into yeast Y2HGold, respectively. Subsequent results promulgated that whether co-transformed with BD-L5CC and AD-T, or BD-P53 and AD-T, yeast transformants could grow normally on DDO medium (SD/-Trp-Leu), but only the latter could grow ordinarily on TDO/X medium (SD/-Trp-Leu-His plus X-α-gal) and displayed the blue-colony phenotype, implying that the bait vector BD-L5 CC did not have self-activation effect (Figure 1).
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Figure 1. BD-L5 CC had no self-activation effect. BD-L5CC and AD-T, as well as BD-P53 and AD-T combinations, co-transform yeast strain Y2HGold competent cell, respectively. Yeast transformants were grown on the DDO media (SD/-Trp-Leu) and on the TDO/X media (SD/-Trp-Leu-His plus X-a-gal). The pair of p53-T used as a positive control. The blue yeast colonies represent positive interactions.
Seven positive colonies that may interact with L5 CC were screened on TDO/X medium through Y2H library screening and the number of repetitions and functional descriptions of each protein in the screening library are shown in Table 1. Functional analysis of these candidate interacting proteins shows that they participate in multiple pathways, including biological and abiotic stress, programmed cell death, protein degradation, material metabolism and transcriptional regulation. PPA1 participated in phosphate-containing compound metabolic process; RIN4 involved in negative regulation of plant-type hypersensitive response; LSU1 participated in cellular oxidant detoxification, cellular response to sulfur starvation and response to salt stress; BZIP24 involved in DNA-templated transcription, negative regulation of response to salt stress, regulation of DNA-templated transcription and regulation of transcription by RNA polymerase II; BOI participated in proteasome-mediated ubiquitin-dependent protein catabolic process and regulation of programmed cell death; as of now, the functionality of RING/U is unknown; PPA3 involved in phosphate-containing compound metabolic process.
Table 1. The functional analysis of AtL5 binding proteins.
Protein name |
Gene locus |
Gene description |
Repeat times |
PPA1 |
AT1G01050 |
Encodes a soluble protein with inorganic pyrophosphatase activity that
is highly specific for Mg-inorganic pyrophosphate. |
4 |
RIN4 |
AT3G25070 |
Encodes a member of the R protein complex and may represent a virulence target of type III pili effector proteins (virulence factors) from bacterial pathogens, which is Primeguard Prime by R protein complex (RPM1
and RPS2 proteins). |
5 |
LSU1 |
AT3G49580 |
RESPONSE TO LOW SULFUR gene family member; expressed during sulfur deficiency. |
4 |
BZIP24 |
AT3G51960 |
bZIP transcription factor induced by salt stress and promoted salt
tolerance. Localized to the cytoplasm and nucleus under control
conditions and targeted preferentially to the nucleus under salt stress. |
3 |
BOI |
AT4G19700 |
Encodes BOI (Botrytis Susceptible 1 Interactor). Has E3 ubiquitin ligase activity. It prevents caspase activation and attenuates cell death. |
6 |
RING/U |
AT4G22250 |
RING/U-box superfamily protein. |
3 |
PPA3 |
AT2G46860 |
Encodes a protein that might have inorganic pyrophosphatase activity. |
4 |
3.2. The Subcellular Localization Analysis of Interaction Proteins of AtL5
L5 receptor is mainly anchored on the PM due to the presence of acylation sites, and PM localization is crucial for the function of the full-length L5 and its CC domain [8]. Our previous articles have demonstrated that the E3 ubiquitin ligase BOI exhibited a nucleocytoplasmic localization and mediated the ubiquitination degradation of L5 protein outside the nucleus [13] [14]. Furthermore, we employed laser confocal microscopy to observe the subcellular localization of six candidate interacting proteins of L5 one by one. Through the Agrobacterium mediated transient expression system in N. benthamiana leaves, we that the fluorescence of RIN4-YFP-HA only appeared on the PM, and RING/U-YFP-HA mainly linked to the PM, while PPA1, PPA3, LSU1, and BZIP24 exhibited nuclear and cytoplasmic distribution in N. benthamiana leaves (Figure 2).
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Figure 2. Observation of subcellular localization of proteins interacting with L5 with confocal microscope. These proteins were transiently expressed in N. benthamiana (OD600 = 0.8). Images were observed at 48 hpi. Scale bars: 25 µm.
3.3. RING/U Can Also Inhibit L5-Mediated Cell Death
The ability of BOI to abolish cell death caused by L5 has been reported [14] (Figure 3(a)). We want to know the impact of the other six candidate proteins that interact with L5 on its function. We fused Myc tag at the C-terminus of these proteins and co-expressed them with L5-YFP-HA into N. benthamiana leaves. The results of transient expression declared that RING/U could also significantly suppress the cell death activity of L5 protein, as shown in Figure 3(b) by trypan blue staining.
(a) (b)
Figure 3. RING/U protein can inhibit cell death triggered by L5 in N. benthamiana. (a) L5-mediated cell death was suppressed by the expression of BOI. L5-YFP-HA was used as a positive control. The photograph was taken at 48 hpi. (b) RING/U protein can also restrain cell death caused by L5. Visible cell death was confirmed by trypan blue staining. Picture was obtained at 48 hpi. Scale bar: 1 cm.
4. Discussion
Currently, multiple Arabidopsis NBS-LRR genes have been cloned, and these proteins may participate in the same or similar signaling pathways to activate downstream defense responses during disease resistance. However, most current research mainly focuses on the identification of disease resistant genes and their interaction with corresponding effectors, with little understanding of their mediated resistance response pathways. This study constructed a high-quality Arabidopsis leaf target cDNA library and screened seven important proteins interacting with L5 using the L5 CC domain as bait (Table 1). Further bioinformatics analysis revealed that the selected candidate proteins include transcription factor, E3 ubiquitin ligases, disease resistance related proteins, and abiotic stress-related proteins.
Transcription factors are participated in various biological processes in plants, comprising pathogen defense, seed maturation, plant growth, flower development, aging, light signaling transduction, damage, and response to various environmental stresses. The interaction between L5 and transcription factor BZIP24 indicates that after recognizing effector, L5 enters the nucleus and binds to transcription factor, activating the immune response through transcriptional regulation, suggesting that L5 protein may undergo repositioning. According to reports, some NBS-LRR proteins, such as MLA10 and RPS4, can be repositioned in the nucleus after activation [11] [12]. Recognition of barley disease resistance protein MLA10 with powdery mildew pathogen effector factor A10 induces MLA10 to bind to transcription factor WRKY in the nucleus to initiate disease resistance response [11].
In Arabidopsis, plasma membrane localized multifunctional protein RIN4 (RPM1interacting protein 4) plays important role in both PTI and ETI [18]. Previous studies have suggested that RIN4 functions as a negative regulator of PTI [19]. Recent study has shown that RIN4 can also promote the extracellular transport of AtEXO70E2 [20]. In addition, many different bacterial effector proteins modify RIN4 to destabilize plant immunity and several NBS-LRR proteins, including RPM1 (resistance to Pseudomonas syringae pv. maculicola 1), RPS2 (resistance to P. syringae 2) guard RIN4 [21] [22]. L5 is a CC-NBS-LRR receptor in Arabidopsis. The activation mechanism of its disease resistance function is still unclear. Considering that L5 can strike cell death in N. benthamiana, we infer an indirect interaction between L5 and the effector. Our previous paper has shown that even if L5-YFP-HA is overexpressed in plants, Arabidopsis did not exhibit an autoimmune phenotype [8]. According to the knowledge of the “guardee model”, there must be interacting proteins in plants that inhibit L5 activation, similar to the relationship with RIN4-RPS2 [22]. Coincidentally, our yeast two hybrid experiment showed that there is also an interaction between L5 and RIN4. Perhaps in addition to RPS2 and RPM1, RIN4 may also be guarded by L5, and this relationship needs further verification in the future.
The level of NBS-LRR protein is under complex control in plants to balance defense responses and other processes. In recent years, the ubiquitination 26S proteasome system has become a key function in regulating NLR stability [23]. In Arabidopsis, it was found that the stability of two NBS-LRRs, SNC1 and RPS2, is controlled by the F-box protein CPR1 (constitutive expression of pathogenesis-related gene 1) [24] [25]. BOI (Botrytis subseptible1 interactor) is a RING type E3 ligase that mediates the ubiquitination of BOS1 (Botrytis subseptible1), a transcription factor involved in stress and pathogen response [26]. BOI inhibits cell death caused by α-picolinic acid (PA), a toxin produced by some fungi that can lead to cell death, as well as virulent pathogens such as Pseudomonas syringae pv. tomato DC3000 (Pto DC3000). Our previous article indicated that BOI degrades the protein level of L5 through ubiquitination, which occurs outside the cell nucleus [13] [14]. Furthermore, in addition to the E3 ubiquitin ligase BOI we previously reported, the new interacting protein RING/U has also been shown to suppress L5-induced cell death in N. benthamiana (Figure 3). In fact, PPA1, PPA3, BZIP24, and LSU1 can all affect the cell death inducing activity of L5 protein to some extent (data not shown), suggesting that the activation of L5 is regulated at multiple levels.
Funding
This study was funded by The Guiding Science and Technology Plan Project of Social Development in Fuzhou City (FKSZ20229003), and The Science and Technology Research Project of Jiangxi Provincial Department of Education (GJJ218112), and The School-level Science and Technology Project of Fuzhou Medical University (fykj202201).
NOTES
*Corresponding author.