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. 2024 Jun 11;4(1):29.
doi: 10.1007/s44154-024-00163-z.

Identification of nuclear membrane SUN proteins and components associated with wheat fungal stress responses

Huan Guo #  1 , Jianfeng Wang #  2 , Di Yao  1 , Ligang Yu  2 , Wenting Jiang  1 , Lincai Xie  1 , Shikai Lv  1 , Xiangyu Zhang  1 , Yajuan Wang  1 , Changyou Wang  1 , Wanquan Ji  1 , Hong Zhang  3   4
Affiliations

Identification of nuclear membrane SUN proteins and components associated with wheat fungal stress responses

Huan Guo et al. Stress Biol. .

Abstract

In eukaryotes, the nuclear membrane that encapsulates genomic DNA is composed of an inner nuclear membrane (INM), an outer nuclear membrane (ONM), and a perinuclear space. SUN proteins located in the INM and KASH proteins in the ONM form the SUN-KASH NM-bridge, which functions as the junction of the nucleocytoplasmic complex junction. Proteins containing the SUN domain showed the highest correlation with differentially accumulated proteins (DAPs) in the wheat response to fungal stress. To understand the characteristics of SUN and its associated proteins in wheat responding to pathogen stress, here we investigated and comprehensive analyzed SUN- and KASH-related proteins among the DAPs under fungi infection based on their conserved motifs. In total, four SUN proteins, one WPP domain-interacting protein (WIP), four WPP domain-interacting tail-anchored proteins (WIT), two WPP proteins and one Ran GTPase activating protein (RanGAP) were identified. Following transient expression of Nicotiana benthamiana, TaSUN2, TaRanGAP2, TaWIT1 and TaWIP1 were identified as nuclear membrane proteins, while TaWPP1 and TaWPP2 were expressed in both the nucleus and cell membrane. RT-qPCR analysis demonstrated that the transcription of TaSUN2, TaRanGAP2 and TaWPP1 were strongly upregulated in response to fungal infection. Furthermore, using the bimolecular fluorescence complementation, the luciferase complementation and a nuclear and split-ubiquitin-based membrane yeast two-hybrid systems, we substantiated the interaction between TaSUN2 and TaWIP1, as well as TaWIP1/WIT1 and TaWPP1/WPP2. Silencing of TaSUN2, TaRanGAP2 and TaWPP1 in wheat leaves promoted powdery mildew infection and hyphal growth, and reduced the expression of TaBRI1, TaBAK1 and Ta14-3-3, indicating that these NM proteins play a positive role in resistance to fungal stress. Our study reveals the characteristics of NM proteins and propose the preliminary construction of SUN-WIP-WPP-RanGAP complex in wheat, which represents a foundation for detail elucidating their functions in wheat in future.

Keywords: Triticum aestivum; Fungal stress; KASH; SUN.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Sequence of SUN and KASH proteins. A Comparison of four SUN domain-containing proteins with AtSUNs. According to the location of the SUN-domain (yellow), TaSUN1 and TaSUN2 are assigned to Cter-SUN, while TaSUN3 and TaSUN4 are assigned to mid-SUN. TaSUN1 contains a signal peptide (purple), and the whole sequence is located in the non-cytoplasmic region (gray). Like Arabidopsis Cter-SUNs, TaSUN2 contains a transmembrane domain (green) a coiled-coil (orange), and a N-terminal in the cytoplasmic region (blue). The structures of mid-SUNs are basically the same, with a SUN-domain in the middle, and three transmembrane domains contained within the whole sequence. B Five novel coiled-coil transmembrane domain proteins interact with WPP domain-containing proteins. The domain structures of the WIT and WIP protein families are similar. The domain structure is characterized by the presence of an extended coiled-coil domain (orange) and a single C-terminal transmembrane domain (green). The AtWIT1 protein does not contain a bipartite nuclear localization signal motif (red), while the coiled-coil domain has not been identified in the SINE protein sequences. The WIP and SINE protein family members contained a predicted cytosolic N-terminal region (blue) and the C-terminal tail in the non-cytoplasmic region (grey), whereas the opposite pattern is observed in the WIT protein family members. TaWIT3 and TaWIT4 are devoid of 1,300 aa and 700 aa, respectively, in the middle of the coiled-coil domains. C Two novel WPP domain-containing proteins. The WPP domain structure is marked in yellow and is characterized by the presence of WPPXXXTR motif. Compared with RanGAP2-like, the WPP proteins are devoid of a C-terminal fragment of the LRR-6 domain (pink) repeat region
Fig. 2
Fig. 2
Transient expression and localization of SUN and KASH proteins. TaSUN1, TaSUN2, TaWIP1, TaWIT1 and TaRanGAP2 localize to the nuclear envelope in N. benthamiana leaf epidermal cells, while TaWPP1 and TaWPP2 localize like free GFP. Free GFP, TaSUN and putative SUN-interacting nuclear envelope proteins fusion construct under the control of the CaMV-35S promoter were transiently expressed into N. benthamiana epidermal cells. Scale bar, 10 µm
Fig. 3
Fig. 3
Interactions between the SUN and KASH proteins in the two-hybrid systems. A Interactions between TaSUN1, TaSUN2, TaWIP1, TaWPP1 and TaWPP2 detected in the nuclear two-hybrid system. pGBKT7-53 and pGBKT7-LAM were used as positive and negative controls, respectively. B MYTH assays between the TaRanGAP2, TaWIP1, TaWIT1 and TaWPP2 as bait and TaSUN2, TaWIP1, TaWPP1 and TaWPP2 as prey. pOst1-NubI and pPR3-N were used as positive control and negative controls, respectively, after co-transfection with the _target gene. Spots corresponding to 100 000 cells on permissive medium (PM) and test medium (TM) are shown. C Tabulated summaries of the strength of protein interactions established based on growth efficiency on solid TM. SD/-Leu/-Trp, double-dropout medium without leucine and tryptophan; QD/x-α-gal, quadruple dropout medium with x-α-gal
Fig. 4
Fig. 4
Interactions between the SUN and KASH proteins in the bimolecular fluorescence complementation (BiFC) assays. The SUN and quasi-interacting protein coding sequences were cloned into nEYFP and cEYFP. After transiently co-transforming N. benthamiana leaf epidermal cells with the nEYFP and cEYFP recombinant vectors, YFP fluorescence confocal micrographs show the location of the interaction. nEYFP and cEYFP empty vectors were co-transfected in N. benthamiana as a negative control, while nEYFP-TaWRKY1-2D and cEYFP-TaDHN3 were employed as a positive control. Scale bars are shown in the lower right corner of each image
Fig. 5
Fig. 5
The activity of TaWIP1, TaWIT1 interacting with both TaSUN2 and TaWPP1 detected by luciferase complementation assay. Photographs of representative leaves were taken 2 d after agroinfiltration. The right pseudocolor bar shows the range of luminescence intensity (e + 05) in each image from high (red) to low (blue)
Fig. 6
Fig. 6
Silencing of TaRanGAP2, TaSUN2, or TaWPP1 reduced resistance to powdery mildew. A Photobleaching of wheat seedlings (four-leaf stage) leaves at 10 dpi with BSMV-PDS; disease phenotype in wheat seedlings (four-leaf stage) leaves inoculated with Bgt E09 at 10 dpi with BSMV-γ. B Representative microscopic images of single colonies of powdery mildew on leaves with monogenic silencing. C RT-qPCR confirmation of decreased expression of TaRanGAP2, TaSUN2, and TaWPP3 in leaves with monogenic silencing. D Colony area of Bgt-infected leaves; data represent the mean ± SD (n = 9 leaves) from three separate experiments. E The average number of total hyphal length of each colony counted on leaves sampled at 24, 48, and 72 hpi of each genotype; data represent the mean ± SD (≥ 50 colonies per genotype) from three independent. **, P < 0.01 (Student’s t-test)
Fig. 7
Fig. 7
Relative transcript levels of TaBRI1, TaBAK1, TaSTK, Ta14-3–3, TaBZR1, and TaPR1 in TaRanGAP2-, TaSUN2- or TaWPP1-slienced plants inoculated with Bgt. Relative gene expression of the silenced line inoculated with Bgt compared with that in control plants at 0 h using Actin as the internal reference. Data represent means ± standard errors (n = 3 biological replicates) of three independent experiments. Differences between time-course sampling points were assessed using SSPS. *P < 0.05
Fig. 8
Fig. 8
Schematic diagram of LINC complex structure. The C-terminal SUN protein in the form of a homotrimer or heterotrimer located in the inner nuclear membrane interacts with WIP and WIT proteins anchored in the outer nuclear membrane to form a heteropolymer. The proteins containing the WPP domain interact with each other, and both interact simultaneously with TaWIT1 and TaWIP1 to anchor the complex to the nuclear membrane. TaRanGAP2 interacts not only with WPP protein, but also with the disease resistance-related proteins BTB/POZ and SKP1
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