Unraveling the Nuclear Localization Sequence of MoHTR2, the Nuclear Effector of Magnaporthe oryzae

Article information

Plant Pathol J. 2025;41(5):583-594

Publication date (electronic) : 2025 September 24

doi : https://doi.org/10.5423/PPJ.OA.06.2025.0080

Jiwon Choi ¹, You-Jin Lim ¹^,²^,

, Yong-Hwan Lee ¹^,²^,³^,

¹Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Korea

²Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea

³Interdisciplinary Program in Agricultural Genomics, Plant Immunity Research Center, Center for Fungal Genetic Resources, and Plant Genomics and Breeding Institute, Seoul National University, Seoul 08826, Korea

^*Co-corresponding authors. Y.-J. Lim Phone) +82-2-880-4684, FAX) +82-2-873-2317 E-mail: dladbwls6297@snu.ac.kr Y.-H. Lee Phone) +82-2-880-4674, FAX) +82-2-873-2317 E-mail: yonglee@snu.ac.kr

Handling Editor : Junhyun Jeon

Received 2025 June 17; Revised 2025 July 23; Accepted 2025 July 28.

Abstract

Plant pathogenic fungi modulate host immunity by secreting nuclear effectors that interact with host nucleic acids and proteins within the host nucleus. Nuclear effectors are widely known to possess a nuclear localization sequence (NLS) that allows them to enter the host nucleus through either the classical importin α-mediated or non-classical pathways. However, the conserved motif in NLS and the mechanism behind successful nuclear trafficking of fungal nuclear effectors remain largely unexplored. MoHTRs, the nuclear effectors of Magnaporthe oryzae, reprogram the transcription of host immunity-associated genes. Recent research has demonstrated that MoHTR1 requires a classical NLS for importin α-mediated entry into the host nucleus and towards the pathogenicity of M. oryzae. However, the NLS of other fungal nuclear effectors, such as MoHTR2, needs further investigation. In this study, we report that MoHTR2 does not interact with rice importin αs or βs. By performing serial truncation and site-directed mutagenesis, we identified ⁵³HH⁵⁴ as the core NLS motif essential for the nuclear localization of MoHTR2. We also found that the double histidine in MGG_13063, a nuclear effector candidate of M. oryzae, is involved in its nuclear localization. Deletion of the MoHTR2 core NLS reduced the invasive hyphal growth and lesion formation by M. oryzae. These findings enhance our understanding of the molecular mechanisms underlying the nuclear localization of fungal nuclear effectors and their roles in pathogenicity, contributing to a broader understanding of host-pathogen interactions.

Keywords: Magnaporthe oryzae; nuclear effector; nuclear localization sequence (NLS); plant fungal pathogen

Plant pathogens prolong their survival by scavenging nutrients from their hosts (Fatima and Senthil-Kumar, 2015). However, host invasion is substantially restricted by the two-tiered immune system of plants. Sessile plants combat infections through recognition of conserved molecular patterns associated with pathogens, leading to the activation of pathogen-associated molecular pattern-triggered immunity (Jones and Dangl, 2006; Nabi et al., 2024). Furthermore, plants use intracellular nucleotide-binding leucine-rich repeat receptors to detect small molecules secreted by pathogens, known as effectors, which consequently initiate effector-triggered immunity (Jones and Dangl, 2006; Nguyen et al., 2021). Effectors are key virulence determinants that can function as enzymes, secondary metabolites, small RNAs or transcription factors and are distinguished by their site of action as apoplastic, cytoplasmic or nuclear effector (Harris et al., 2023; Rocafort et al., 2020; Toruño et al., 2016).

Nuclear effectors modify host processes through diverse mechanisms, including epigenetic regulation, RNA interference and modulation of immunity-associated genes (Harris et al., 2023). While small proteins, typically less than 40 kDa, can pass through the nuclear pore complex (NPC) via passive diffusion, larger proteins require a nuclear localization sequence (NLS) to facilitate their transport into the nucleus (Sun et al., 2016). NLS is a short peptide signal and can be categorized into classical (cNLS) or non-classical (ncNLS) based on their structural attributes (Harris et al., 2023). cNLS can be further divided into monopartite, comprising a short stretch of basic amino acids, particularly arginine (R) and lysine (K) or bipartite, harbouring two clusters of positively charged amino acids separated by 10–20 amino acid residues (Lu et al., 2021). On the other hand, ncNLS does not have a confined structure and is not necessarily rich in arginine (R) or lysine (K). The most well-understood ncNLS is PY-NLS, which ends with a proline (P)-tyrosine (Y) motif. Furthermore, there are special NLSs, such as the tripartite NLS, which consists of three clusters of basic amino acids, and the cryptic NLS, which is only exposed if post-translational modifications have occurred (De Mandal and Jeon, 2022; Escoto et al., 2024; Mattola et al., 2022).

Since the discovery of the first monopartite NLS, Simian Virus 40 T antigen NLS (PKKKRKV) (Kalderon et al., 1984), the mechanisms underlying the nuclear transport of nuclear effectors have been extensively studied. Nuclear effectors carrying a cNLS interact directly with importin α, which subsequently recruits importin β1 to mediate nuclear import (Kosugi et al., 2009). For example, PvAVH53, a nuclear targeting RxLR effector of the Oomycete Plasmopara viticola, binds with the host importin α1 and importin α2 to reach the nucleus in Nicotiana benthamiana (Chen et al., 2021). Similarly, PthXo1, a TAL effector of plant pathogenic bacterium Xanthomonas oryzae pv. oryzae (Xoo) requires importin α1b to translocate into the host nucleus and harness the rice defense response (Peng et al., 2022). Compared to the classical pathway, the understanding of non-classical transport mechanisms of nuclear effectors remains incomplete.

Magnaporthe oryzae is a hemibiotrophic fungal pathogen that causes blast disease on cereal crops, including rice and wheat. The blast disease is prevalent worldwide, covering all rice-growing countries and can lead to 30% rice yield loss, posing an enormous threat to global rice production (Batool et al., 2024; Zhang et al., 2022). During the biotrophic infection stage, M. oryzae secretes nuclear effectors, MoHTRs (Magnaporthe oryzae Host Transcriptional Reprogramming), from the biotrophic interfacial complex (BIC) to hamper the host immune response (Kim et al., 2020). MoHTR1 and MoHTR2 suppress the expression of OsMYB4, OsWRKY45, and OsHPL2, which are responsible for rice defense response (Kim et al., 2020). Previous research revealed that MoHTR1 possesses a core NLS (RxKK) and that its interaction with rice importin αs is required for translocation to the host nucleus and transcriptional reprogramming of immunity-related genes (Lim et al., 2024). However, the nuclear transport mechanism of other fungal nuclear effectors remains unknown.

In this work, we confirmed that MoHTR2 does not directly interact with rice importin αs and importin βs to pass through the NPC. We identified the core NLS of MoHTR2, which is distinct from that of MoHTR1 and found it essential for nuclear trafficking. Additionally, we validated that the double histidine also plays an important role in another nuclear effector candidate of M. oryzae, MGG_13063. Moreover, we verified that double histidine in MoHTR2 holds an essential role in the pathogenicity of M. oryzae. Our findings highlight the importance of the core NLS in the nuclear trafficking and pathogenicity of M. oryzae nuclear effectors.

Materials and Methods

Plasmid preparation for rice protoplast transfection

Different constructs of MoHTR2 without signal peptide (MoHTR2Δsp) and MGG_13063Δsp were PCR amplified from the cDNA of the wild-type strain (KJ201) of M. oryzae using the following primers (Supplementary Table 1) and Platinum SuperFi II Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). The amplified constructs were purified using the MEGAquick-spin Plus Total Fragment DNA Purification Kit (iNtRON Biotechnology, Seongnam, Korea). For transient expression in rice protoplasts, the purified constructs were first cloned into the pENTR/D-TOPO gateway vector (Thermo Fisher Scientific) and then reintroduced into the final eGFP expression vector p2GWF7 (Karimi et al., 2002) using the Gateway LR Clonase II Enzyme Mix (Invitrogen, Carlsbad, CA, USA). Before the protoplast transfection assay, the final plasmids were prepared using the PureLink HiPure Plasmid Midiprep Kit (Invitrogen).

Rice protoplast transfection and observation

Peeled rice seeds (Oryza sativa cv. Nakdongbyeo) were surface-sterilized using 70% ethanol and 2% NaOCl. We planted 25–30 sterilized seeds in 100 mL 1/2 MS medium and incubated them in a dark chamber at 25°C for 10–14 days. We then isolated rice protoplasts using a previously described method (Lim et al., 2024; Zhang et al., 2011). We finely chopped the 10–14-day-old rice tissues and incubated with lysing enzyme solution (containing 1.5% cellulase and 0.75% Macerozyme R-10) for 3 h to extract protoplast of 2 × 10⁶ cells/mL. For a transient expression of plasmid, we used the PEG-mediated transfection method by adding 5 μg of eGFP tagged MoHTR2 variants and 5 μg of nucleus marker, OsABF1::mRFP in 100 μL of rice protoplast. After 16 h, we observed at least 120 rice protoplasts per experiment under a fluorescence microscope (Leica DM6000B, Leica Microsystems, Wetzlar, Germany).

Site-directed mutagenesis

To find the NLS and the core motif in NLS of MoHTR2, we performed site-directed mutagenesis using Golden mutagenesis (Püllmann et al., 2019), QuickChange Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA, USA), and GeneArt Site-Directed Mutagenesis PLUS System (Thermo Fisher Scientific), following the manufacturer’s instruction. The primers used for the individual site-directed mutagenesis are provided in the supplementary data (Supplementary Table 1).

Yeast two-hybrid (Y2H) assay

For the bait, pENTR/D-TOPO::MoHTR2Δsp was cloned to pDEST32, respectively. For the prey, three rice importin αs and three rice importin βs, selected from the Rice Genome Annotation Project Database (https://rice.uga.edu/) (Peng et al., 2022), were used. OsImpα (Os01g24060), OsImpα1a (Os01g14950), OsImpα1b (Os05g06350), OsImpβ_1 (Os03g18350), OsImpβ_2 (Os12g38110), and OsImpβ1 (Os05g28510) were PCR amplified from the rice cDNA, and were cloned to pENTR/D-TOPO, then finally LR reacted to pDEST22. The gateway recombination reactions were performed using Gateway LR Clonase II Enzyme Mix (Invitrogen). The bait and prey plasmids were co-transformed into the yeast strain MaV203, following the manual provided by ProQuest Two-Hybrid System (Invitrogen). The transformation was confirmed by growing the transformed yeast on SC-Leu-Trp (SC-LT) agar media at 30°C for 3 days. A single colony was selected and regrown in YPAD broth in the shaking incubator at 30°C, 150 rpm, overnight, then washed twice with 0.9% NaCl. The protein-protein interaction was tested on SC-Leu-Trp-His + 25 mM 3-amino-1,2,4-triazole (SC-LTH + 3-AT) agar plate via serial drop inoculation.

Selection of nuclear effector candidates having HH in their predicted NLS

Out of the 1,899 proteins known to be secreted by M. oryzae, according to the Fungal Secretome Database, we previously identified 440 proteins with predicted NLS using three NLS prediction tools: WoLF PSORT, cNLS mapper, and NLStradamus (Lim et al., 2024). Among these, we identified five proteins with an ‘HH’ motif in their predicted NLS. Of these five proteins, we successfully amplified three protein encoding genes from KJ201 cDNA and performed quantitative reverse transcription polymerase chain reaction (qRT-PCR) to examine effector characteristics.

Fungal genomic DNA extraction

KJ201 mycelia were cultured in CM broth at 25°C in a shaking incubator set at 150 rpm for 3 days. The collected mycelia were freeze-dried and ground into a fine powder using the FastPrep-24TM 5G homogenizer (MP Biomedicals, Irvine, CA, USA). The powdered sample was treated with genomic DNA (gDNA) extraction buffer (100 mM EDTA, 100 mM Tris-HCl, 250 mM NaCl, 100 μg/mL RNaseA, and 100 μg/mL proteinase K). The mixture was incubated at 50°C for 1 h, followed by adding 10% N-lauroylsarcosin and a second incubation at 55°C for 1 h. The lysate was centrifuged, and the supernatant was treated with 1:1 phenol:chloroform solution, followed by incubation on a rocking shaker at 15 rpm for 15 min. After a second centrifugation, the supernatant was treated with a 24:1 chloroform: isoamyl alcohol solution. Following a third centrifugation, the pellet was washed sequentially with isopropanol and ethanol and then eluted with water.

ΔMohtr2::MoHTR2^ΔIHH generation

To generate ΔMohtr2:: MoHTR2^ΔIHH, we first generated ΔMohtr2 protoplast. The knockout strain, ΔMohtr2, was obtained from the previous study (Kim et al., 2020). We grew ΔMohtr2 in a complete medium broth for 3 days in the shaking incubator at 25°C, 200 rpm. Afterwards, we treated the mycelia with CW-lytic F1 (Amicogen, Jinju, Korea) and washed it with 2× STC buffer using the previously described method (Lee et al., 2023). To make the MoHTR2^ΔIHH construct, we extracted the gDNA from KJ201 using the protocol mentioned above. By using KJ201 gDNA as a template, we amplified the upstream and downstream of MoHTR2 ORF, which excludes the protein sequence ‘IHH’ in the middle, using Platinum SuperFi II Green PCR Master Mix (Thermo Fisher Scientific) and Phusion Hi-Fi DNA polymerase (Invitrogen). The geneticin cassette from PII99 (Namiki et al., 2001) was used as a selection marker. Before use, all constructs were purified with MEGAquick-spin Plus Total Fragment DNA Purification Kit (iNtRON Biotechnology). The prepared construct and geneticin cassette were introduced into ΔMohtr2 protoplast using the PEG-mediated transformation method. The complemented strains were selected from a TB3 agar plate containing 800 ppm geneticin.

Pathogenicity test

For sheath inoculation, KJ201, ΔMohtr2, and ΔMohtr2::MoHTR2^ΔIHH were each grown on V8 agar for 6 days. 2 × 10⁴/mL conidial suspension was applied to the sheath isolated from 6-week-old Nakdongbyeo. At 48 hour post-inoculation (hpi), we observed invasive hyphal growth at least 120 infection sites per experiment under a microscope (Leica DM6000B). For spray inoculation, KJ201, ΔMohtr2 and ΔMohtr2::MoHTR2^ΔIHH were each grown on oatmeal agar for 10 days. 10⁵/mL conidial suspension with 250 ppm Tween 20 (Bio-Rad, Hercules, CA, USA) was sprayed onto 4-week-old Nakdongbyeo and kept in a dark dew chamber with 100% humidity overnight. The inoculated Nakdongbyeo was further grown in a plant growth chamber under 25°C, 70% humidity. At 6 days post-inoculation (dpi), we collected inoculated leaves and the diseased area was calculated using ImageJ (Schneider et al., 2012).

RNA extraction and qRT-PCR

We extracted RNA from mycelia and from rice infected with wild type, ΔMohtr2, and ΔMohtr2::MoHTR2^ΔIHH using the easy-spin Total RNA Extraction Kit (iNtRON Biotechnology), following the manufacturer's instructions. After confirming the purity of extracted RNA using Nanodrop 1000 (Thermo Fisher Scientific), we synthesized cDNA using the ImProm-II Reverse Transcription system (Promega, Madison, WI, USA). Each qRT-PCR reaction containing 2× Rotor-Gene SYBR Green PCR master mix (Qiagen, Hilden, Germany), 25 ng cDNA and 15 pmol of forward and reverse primer (Supplementary Table 1) was run in Rotor-Gene Q real-time PCR cycler (Qiagen). The gene expression level was quantified using the Delta-Delta Ct method (Livak and Schmittgen, 2001).

Results

MoHTR2 does not interact with rice importins

Proteins with cNLSs are known to bind with importin α for nuclear import. The importin-mediated pathway is the most extensively studied mechanism for delivering NLS-carrying proteins into the nucleus (King et al., 2020). A previous study demonstrated that MoHTR1 interacts with rice importin αs for nuclear localization (Lim et al., 2024). To investigate whether MoHTR2 also utilizes the same nuclear localization transport mechanism, we performed a yeast two-hybrid assay to test its interactions with rice importin αs. We used MoHTR2 as bait and importin subunits as prey. The results showed that serially diluted yeast transformants failed to grow on SC-LTH + 25 mM 3-AT medium, indicating that MoHTR2 does not interact with the importin α subunits (OsImpα, OsImpα1a, and OsImpα1b) in vitro (Fig. 1A). Some NLS-carrying proteins are known to directly interact with importin β, without the presence of importin α, for nuclear localization (Cingolani et al., 2002). Therefore, we further examined the interaction between MoHTR2 and three rice importin βs (OsImpβ_1, OsImpβ_2, and OsImpβ1) in vitro. None of importin β subunits interacted with MoHTR2 (Fig. 1B). These findings suggest that MoHTR2 does not rely on classical importin α-mediated pathways for nuclear localization, unlike MoHTR1.

Fig. 1

Interaction of MoHTR2 with rice importins. Yeast two-hybrid (Y2H) assays were performed to evaluate the interaction between MoHTR2 (MoHTR2Δsp) and rice importins. pDEST22:: MoHTR2 and pDEST32::OsImps were co-transformed into the yeast strain Mav203. Protein-protein interactions were evaluated by dropping serial dilutions of yeast cultures on selective media, SC-LT and SC-LTH + 25 mM 3-AT. For the positive control, the pEXP32::Krev1 and pEXP22::RalGDS-wt were co-transformed. For the negative control, pEXP32::Krev1 and pEXP22::RalGDS-m2 were co-transformed. (A) Interaction of MoHTR2 with rice importin α1, importin α1a, and importin α1b. (B) Interaction of MoHTR2 with rice importinβ_1, importinβ_2 or importin β1. Similar results were obtained across three independent experiments. The figure presents representative data from one of these experiments.

MoHTR2 contains an NLS in the N-terminus

MoHTR2 is a 110-amino-acid (a.a.) protein comprising an N-terminal signal peptide and a C₂H₂ zinc finger domain (residues 68–101). A predicted NLS, LKPSKG, is located within the zinc finger domain, as identified by the NLS prediction tool, cNLS mapper (Fig. 2A). To understand whether this predicted sequence functions as a true NLS, we truncated MoHTR2Δsp into N-terminal (19–63 a.a.) and C-terminal (64–110 a.a.) segments, tagged each with eGFP, and compared their localization in rice protoplasts. The transfection assay reveals that MoHTR2_N localizes in the nucleus in 59 % of transfected rice protoplasts, comparable to the nuclear localization of full-length MoHTR2, which is also 59%. In contrast, MoHTR2_C, which contains the predicted NLS, displayed a much reduced nuclear localization of 35% (Fig. 2B and C). Having confirmed that the predicted NLS of MoHTR2 does not function as a true NLS, we performed serial truncations by adding fragments of MoHTR2_N to MoHTR2_C to identify the location of a potential NLS. After adding the last 15 amino acids of MoHTR2_N (49–63 a.a.), shown as MoHTR2_C_2, we observed 60% nuclear localization, an increased nuclear localization compared to MoHTR2_C (35%). To further confirm that no additional amino acids are required for nuclear transport, we added the last 30 amino acids of MoHTR2_N and examined the localization. MoHTR2_C_1 (34–110 a.a.) has 58% nuclear localization. Taken together, our data indicate that the predicted NLS of MoHTR2 does not function as a true NLS. Instead, the functional NLS is likely located near the N-terminus, between 49–63 a.a.

Fig. 2

Nuclear localization of serially truncated MoHTR2 in rice protoplasts. (A) Schematic representation of MoHTR2. MoHTR2 has predicted nuclear localization sequence (NLS) within the C₂H₂ zinc finger domain. (B) Rice protoplast transfection assays were conducted to examine the nuclear localization of serially truncated MoHTR2Δsp constructs tagged with eGFP. The rice transcription factor tagged with mRFP, OsABF1::mRFP, was used as a nuclear marker and double eGFP was used as a negative control. Fluorescence microscopy images of rice protoplasts transfected with serially truncated MoHTR2 variants. Scale bars = 10 μm. (C) The percentage of rice protoplasts displaying nuclear localization of eGFP-tagged MoHTR2 variants (mean ± standard deviation; n = 3). Three independent experiments demonstrated similar results, with representative data shown in this figure. Unpaired Student’s t-test was performed, and the significance is indicated by asterisks (*P < 0.05, **P < 0.01, and ***P < 0.001).

Double histidine (HH) serves as the core NLS of MoHTR2

Basic amino acids such as lysine (K), arginine (R), and histidine (H) are known to play essential roles in NLS (Chaudhary et al., 2014). To investigate their role in the potential NLS of MoHTR2, we substituted three arginine residues (R56, R60, and R63) and two histidine residues (H53 and H54) with alanine (A) and observed changes in nuclear localization. The mutation of the three arginines, MoHTR2_R-A, did not affect the nuclear localization of MoHTR2. However, the additional mutation of the two histidine residues, MoHTR2_H, R-A, reduced nuclear localization to 32% (Fig. 3A and B). To further confirm the roles of the double histidine in the NLS of MoHTR2, we performed serial site-directed mutagenesis of 49–63 a.a. in MoHTR2 by sequentially replacing three amino acids with three alanine residues. The mutation of the first three amino acids, MoHTR2_3aa-A, caused no significant change in nuclear localization compared to MoHTR2 by having 66% nuclear localization. However, with the mutation of three additional amino acids containing the double histidine, MoHTR2_6aa-A, nuclear localization decreased to 43%, similar to the level of MoHTR2_C (33%) (Fig. 4A and B). To consolidate the core NLS, we further observed the nuclear localization of MoHTR2_9aa-A. Three extra mutations had similar levels of nuclear localization to MoHTR2_3aa-A. Therefore, these findings suggest that ⁵³HH⁵⁴ is the core NLS of MoHTR2.

Fig. 3

Site-directed mutagenesis of basic amino acids in the potential nuclear localization sequence (NLS) of MoHTR2. Rice protoplast transfection assays were performed to investigate the role of basic amino acids in the potential NLS region of MoHTR2. The rice transcription factor tagged with mRFP, OsABF1::mRFP, was used as a nuclear marker and double eGFP was used as a negative control. (A) Fluorescence microscopy images of rice protoplasts transfected with arginine and histidine mutant of MoHTR2Δsp. Scale bars = 10 μm. (B) The percentage of cells eGFP tagged constructs localized in the rice nucleus (mean ± standard deviation; n = 3). Three independent experiments demonstrated similar results, and the data represent one independent experiment. Unpaired Student’s t-test was performed, and the significance is indicated by asterisks (*P < 0.05, **P < 0.01, and ***P < 0.001).

Fig. 4

Nuclear localization of serially mutated MoHTR2 in rice protoplasts. Rice protoplast transfection assays were performed to examine the localization of serially mutated MoHTR2Δsp constructs tagged with eGFP. The rice transcription factor tagged with mRFP, OsABF1::mRFP, was used as a nuclear marker and double eGFP was used as a negative control. (A) Fluorescence microscopy images of rice protoplasts transfected with serially mutated MoHTR2 variants. Scale bars = 10 μm. (B) The percentage of rice protoplasts displaying nuclear localization of eGFP-tagged MoHTR2 variants (mean ± standard deviation; n = 3). Three independent experiments demonstrated similar results, with representative data shown in this figure. Unpaired Student’s t-test was performed, and the significance is indicated by asterisks (*P < 0.05, **P < 0.01, and ***P < 0.001).

Double histidine is required for the nuclear localization of another nuclear effector candidate, MGG_13063

The roles of lysine (K) and arginine (R) in NLS have been studied in depth, as they are common basic amino acids found in cNLS motifs (Lu et al., 2021). However, the role of histidine (H) in NLS function has not been thoroughly investigated. To address this gap and investigate the possible role of double histidine in NLS of other nuclear effectors, we identified M. oryzae nuclear effector candidates that contain a double histidine within its predicted NLS from the Fungal Secretome Database using NLS prediction tools (Supplementary Table 2). Among the HH-containing effector candidates, we revealed that MGG_13063 exhibits a significant increase in expression during the infection stage by performing qRT-PCR (Fig. 5A). MGG_13063 has two predicted NLSs with double histidine within the NLS predicted by NLStradamus and WoLF PSORT (Fig. 5B). Furthermore, it predominantly localizes in the rice nucleus, showing 63% nuclear localization (Fig. 5C and D). To determine whether the double histidine contributes to the nuclear trafficking of MGG_13063, we performed site-directed mutagenesis. Mutation of the two histidines (H277 and H278) reduced nuclear localization to 39% (Fig. 5C and D). These results established that double histidine is required for the nuclear localization of MGG_13063, implying that double histidine also plays an important role in the NLS of other fungal nuclear effectors.

Fig. 5

The importance of double histidine (HH) in the nuclear effector candidate MGG_13063 for nuclear localization. (A) Expression of MGG_13063 in fungal mycelia and during 3 infection stages (24 hour post-inoculation [hpi], 48 hpi, and 72 hpi). Fold changes were calculated using the 2^−ΔΔCt method (mean ± standard deviation [SD]; n = 3). Three independent experiments demonstrated similar results, and the representative data of one experiment is shown. Paired Student’s t-test was performed, and it is shown by asterisks (*P < 0.05, **P < 0.01, and ***P < 0.001). (B) Schematic representation of MGG_13063 and its double histidine mutant, MGG_13063_HH-A. Predicted nuclear localization sequences (NLSs), identified using NLStradamus and WoLF PSORT, are highlighted in yellow and green boxes, respectively. (C) Fluorescence microscopy images of rice protoplasts transfected with MGG_13063 and its double histidine mutant, MGG_13063_HH-A. The rice transcription factor tagged with mRFP, OsABF1::mRFP, was used as a nuclear marker and double eGFP was used as a negative control. Scale bars = 10 μm. (D) Percentage of cells expressing eGFP tagged constructs in the rice nucleus (mean ± SD; n = 3). Three independent experiments demonstrated similar results, and the data represent one independent experiment. Unpaired Student’s t-test was performed, and it is shown by asterisks (*P < 0.05, **P < 0.01, and ***P < 0.001).

MoHTR2 core NLS is crucial for the pathogenicity of M. oryzae

We conducted pathogenicity assays on rice to further investigate the role of MoHTR2’s core NLS. First, we inoculated 6-week-old rice sheath cells with 2 × 10⁴/mL conidial suspension of the wild type, ΔMohtr2, and two independent ΔMohtr2::MoHTR2^ΔIHH strains. After 48 hpi, we classified the infected cells into four disease types based on the degree of invasive hyphal growth originating from the initially penetrated cells. A previous study highlighted that ΔMohtr2 exhibits decreased invasive hyphal growth compared to the wild type (Kim et al., 2020). Similarly, we observed reduced pathogenicity of ΔMohtr2 compared to the wild type, as indicated by 38% of Type I and 52% of Type II cells for the wild type and 60% of Type I and 34% of Type II cells for the ΔMohtr2. Upon inoculation with the two independent ΔMohtr2::MoHTR2^ΔIHH strains, we found 59% of Type I cells and 33–35% of Type II cells, indicating fewer Type II cells and more Type I cells compared to the wild type that is similar to ΔMohtr2 (Fig. 6A). To further examine pathogenicity, we performed spray inoculation by applying 10⁵/mL conidial suspension onto 4-week-old rice seedlings and measured the diseased area 6 dpi. The wild type exhibited a diseased area of 27%, and ΔMohtr2 displayed a reduced diseased area of 14%, showing a trend consistent with previous findings (Kim et al., 2020). In our work, similar to ΔMohtr2, ΔMohtr2::MoHTR2^ΔIHH demonstrated a reduced percentage of diseased area (11%) compared to the wild type (Fig. 6B). Since the ΔMohtr2::MoHTR2^ΔIHH strain exhibited markedly reduced pathogenicity compared to the wild type, we assessed the expression levels of pathogenesis-related (PR) genes in rice, PR1a, PR1b, PR2, and PR10a, previously reported to be upregulated in ΔMohtr2-infected rice (Kim et al., 2020). Expression of these PR genes was significantly increased not only in ΔMohtr2-infected rice but also in ΔMohtr2::MoHTR2^ΔIHH-infected rice compared to the infected rice with wild type (Fig. 6C). These results underscore that the double histidine motif, the core NLS of MoHTR2, affects the pathogenicity of M. oryzae.

Fig. 6

The role of double histidine in MoHTR2 towards M. oryzae pathogenicity. (A) Rice sheaths were inoculated with 2 × 10⁴/mL conidial suspensions of the wild type, ΔMohtr2, and two ΔMohtr2::MoHTR2^ΔIHH (ΔMohtr2::MoHTR2^ΔIHH_1 and ΔMohtr2::MoHTR2^ΔIHH_2) strains. Invasive hyphal growth was classified into four categories (Type I–IV) at 48 hour post-inoculation (hpi) based on the extent of invasive hyphal growth. Type I, invasive hyphal growth restricted primarily to the initially invaded cell; Type II, invasive hyphal growth extended to the neighboring cell; Type III, invasive hyphal growth reached the third cell from the initially infected cell; Type IV, invasive hyphal growth spread to more than four cells starting from the initially invaded cell. The representative phenotype of each type is shown as a microscopy image, with a scale bars = 20 μm. The percentage of each type of invasive hyphal growth is represented as a stacked bar graph (mean ± standard deviation [SD]; n = 3). (B) 4-week-old rice seedlings were sprayed with 10⁵/mL conidial suspensions of wild type, ΔMohtr2, and ΔMohtr2::MoHTR2^ΔIHH. At 6 dpi, the inoculated leaves were collected, and the percentage of the diseased leaf area was calculated using ImageJ (mean ± SD; n = 3). Three independent experiments produced similar results, and the data presented represent one of three experiments. Student’s t-test was performed, and it is shown by asterisks (*P < 0.05, **P < 0.01, and ***P < 0.001). (C) Transcription levels of pathogenesis-related (PR) genes in wild type, ΔMohtr2, and ΔMohtr2::MoHTR2^ΔIHH-infected rice. Infected rice leaves were collected at 48 hpi following spray inoculation, and total RNA was extracted from each sample. Three independent experiments produced similar results, and the data presented represent one of three experiments. Student’s t-test was performed, and it is shown by asterisks (***P < 0.001).

Discussion

Nuclear effectors of plant pathogens modulate multiple nuclear processes that are required for the host immunity. In the biotrophic infection stage, M. oryzae secretes the nuclear effectors (MoHTR1 and MoHTR2) from the BIC to interfere with the host transcription of immunity-associated genes and successfully invade the host (Kim et al., 2020; Lee et al., 2023). Previous research identified RxKK, a cNLS, as the essential sequence required for the nuclear transport of MoHTR1 via an importin α- and post-translational modification-mediated pathway (Lim et al., 2024). However, the nuclear transport mechanisms of other fungal nuclear effectors remain largely unexplored.

Nuclear effectors secreted by the same species sometimes share the same NLS. The bacterial TAL effector PthXo1 of Xoo and TALI of X. oryzae pv. oryzicola both contain identical NLSs (KRAKP, RKRSR, and WRVKRPRTR) within the C-terminus (Peng et al., 2022). Similarly, the CRN effectors of Phytophthora sojae, PsCRN63, PsCRN77, PsCRN84, and PsCRN86, carry the same NLS (QRKRYRR) (Liu et al., 2011). However, our findings demonstrate that nuclear effectors from the same species can rely on distinct core NLSs for specific targeting of the host nucleus. Each MoHTR effector possesses a unique core NLS despite all being secreted from the BIC during the biotrophic infection stage to modulate immunity-related genes. Our results emphasize the diversity of nuclear localization strategies among nuclear effectors, highlighting that while some species utilize shared NLS motifs for host nucleus targeting, M. oryzae nuclear effectors employ distinct core NLSs to achieve specialized and multifaceted reprogramming of host immunity.

Apart from MoHTR1, many nuclear effectors of other plant pathogens also utilize an importin α-mediated pathway for delivering nuclear effectors into the nucleus. For instance, the TAL effector PthA variants 1–4 of the plant pathogenic bacterium Xanthomonas citri all interact with importin α to facilitate nuclear entry (De Souza et al., 2012). Similarly, CRN effectors such as CRN2, CRN8, and CRN15 from the Oomycete Phytophthora infestans require host importin α to accumulate in the host nucleus (Schornack et al., 2010). In other cases, some nuclear proteins carrying ncNLS are known to interact with importin βs for nuclear transport. For instance, the Arabidopsis growth regulatory protein GIF1 interacts with importin β4 to facilitate nuclear accumulation (Liu et al., 2019). However, in this study, we verified through a Y2H assay that MoHTR2 does not interact with 3 rice importin α and 3 importin β subunits. Beyond the examined rice importins, rice encodes additional importins such as importin 7 and importin 8 (Peng et al., 2022). These importins have established roles in human cells. For example, importin 7 mediates the nuclear transport of MEK1, a serine-threonine kinase, in human CD4 T cells (Panagiotopoulos et al., 2021), and importin 8 facilitates the nuclear import of eIF4E, a transcription factor, in human cells (Guo et al., 2013). We can not entirely exclude the possibility that rice importins 7 or 8 in rice could contribute to the nuclear delivery of MoHTR2. Another possibility would be engaging with non-importin rice proteins to traverse the NPC like LntA, a bacterial nuclear effector of Listeria monocytogenes (Lebreton et al., 2014). All together, our findings suggest that MoHTR2 exploits non-classical pathways, distinct from MoHTR1, to traverse the NPC, providing insight into the diverse nuclear transport mechanisms the fungal nuclear effectors utilize.

We identified that the double histidine (⁵³HH⁵⁴) is the core NLS of MoHTR2. Without the core NLS, MoHTR2 failed to completely localize in the rice nucleus and exert pathogenic effects, which were demonstrated by a decrease in invasive hyphal growth and lesion formation. Until now, investigations on nuclear effector NLS mostly focused on lysine (K) and arginine (R) compared to histidine (H). However, histidine (H) has also been shown to play a key role in the nuclear proteins of various organisms. For instance, N. benthamiana OpsDHN12 has a histidine-rich motif in its NLS that is required for the nuclear localization (Hernández-Sánchez et al., 2015). In addition, BFLF2, the nuclear egress protein of the human Epstein-Barr virus, requires H71 and H74 for nuclear localization (Dai et al., 2020). We further demonstrated a significant role of double histidine for nuclear localization in another nuclear candidate, MGG_13063, of M. oryzae. Mutation of the ²⁷⁷HH²⁷⁸ motif reduced nuclear localization. Our work expands this framework by implicating histidine as a key player in nuclear trafficking, particularly in fungal effectors, for the first time.

Our study demonstrated that MoHTR2 relies on distinct, non-classical core motifs for nuclear targeting that differs from the importin α-mediated pathway utilized by MoHTR1. The identification of the double histidine (HH) motif as a key determinant in nuclear localization provides the first evidence of its role in fungal nuclear effectors beyond lysine and arginine residues. The discovery of the HH motif expands the known repertoire of nuclear trafficking signals in plant-pathogen interactions and raises new questions about how non-canonical NLSs are recognized by host nuclear transport machinery. This insight offers a novel perspective for identifying nuclear effectors that exploit unconventional nuclear translocation mechanisms and provides a valuable framework for exploring how core NLS contribute to pathogenicity and the manipulation of host immunity.

Notes

Conflicts of Interest

No potential conflict of interest relevant to this article was reported.

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grants funded by Ministry of Science and ICT (MSIT) (RS-2023-00275965 and RS-2025-00512558 to Y.-H.L. and RS-2023-00246565 to Y.-J.L.). J.C. is grateful for a Brain Korea 21 Plus Program graduate fellowship.

Electronic Supplementary Material

Supplementary materials are available at The Plant Pathology Journal website (http://www.ppjonline.org/).

PPJ-OA-06-2025-0080-Supplementary-Table.pdf

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