Involvement of the Transporter CgTrk1 in Potassium Uptake, Invasive Growth, and Full Virulence in Colletotrichum gloeosporioides

: Colletotrichum gloeosporioides is one of the more economically important pathogen causing anthracnose on crops and trees worldwide. As an essential mineral nutrient, potassium play a vital role for fundamental cellular processes in organisms. In this study, a high-affinity potassium transporter CgTrk1 was identified in C. gloeosporioides . Cytological examinations revealed that CgTrk1 was localized in the plasma membrane. The gene deletion mutant of CgTRK1 significantly depressed the potassium uptake. CgTrk1 is also required for vegetative growth, appressorium development, invasive growth, and full virulence. The data also indicate that CgTrk1 plays dominant roles in potassium uptake and pathogenicity rather than its homologous protein CgTrk2. These results imply that the potassium transporter CgTrk1 is involved in invasive growth and full virulence in C. gloeosporioides.


Introduction
Potassium (K + ) is an essential mineral nutrient for organisms to fulfill multiple functions, including keeping intracellular monovalent cation homeostasis and transmembrane electrical potential, maintaining cellular turgor pressure and morphology, and regulating fundamentally and sophisticated signaling networks [1][2][3].However, there is a significantly asymmetric distribution between intracellular and extracellular potassium concentrations.Cells acquire potassium against a concentration gradient into the cell through active and passive mechanisms [4].In Saccharomyces cerevisiae, a variety of potassium transporters have been confirmed to facilitate potassium uptake, such as the high-affinity K + transporter Trk protein family [2,5].The conserved Trk protein family has been identified in fungi, bacteria, and plants, which mediates K + influx via the membrane potential generated by the H + -ATPase [5,6].Trk1 consists of 12 potential transmembrane (TM) domains.Four monomers of Trk1 form a tetramer with a central pore and are embedded in the cell membrane in S. cerevisiae [7][8][9].Trk2 encodes 889 amino acids shows a high affinity to Trk1 [10,11].Mutants lacking TRK1 and/or TRK2 result in a lower potassium content, higher sensitivity to sodium and acid environments, and hyperpolarization of the plasma membrane in fission yeasts [12].Cells have evolved a complex regulation system to manipulate the activity of potassium transport.It has been reported that a larger number of proteins directly or indirectly regulate Trk transporters in the stages of transcription, post-translation, or subcellular localization [13,14].In S. cerevisiae, the activity of Trk1 depends on its phosphorylation balance mediated by the kinase Hal4/Hal5 and the phosphatase Ppz1/Ppz2 [15,16].The Ppz1 reduces potassium influx by regulating the high-affinity Trk transporter system, which contributes to cellular turgor pressure and resistance against cell wall stressors [17,18].In C. gloeosporioides, CgPpz1 is also involved in potassium influx and responds to extracellular cation stress [19].The protein kinase Sat4 positively phosphorylates Trk1 to regulate its subcellular localization rather than expression [14,20].However, the mechanism of Trk transporter regulating potassium uptake and plant infection in phytopathogenic fungi is not completely understood.
Anthracnose caused by C. gloeosporioides leads to a large number of economic losses on crops, fruits, and trees, such as Brassica chinensis, bananas, mangoes, and Cunninghamia lanceolata [21,22].C. gloeosporiodes typically develops specialized structures, including appressoria and invasive hyphae, to infect host plants [21].Potassium plays a crucial role in fulfilling the nutritional and chemiosmotic requirements of phytopathogens, which helps to modulate their resistance to antimicrobials by mediating the expression of virulence factors [3,23,24].Potassium transporters have been required for hyperosmotic tolerance, acid stress resistance, colonization, and virulence in Listeria monocytogenes and Streptococcus mutans [25][26][27][28].The homologous protein kinases ChSat4 and CgSat4 facilitate the accumulation of intracellular potassium by regulating the subcellular localization of Trk1.Meanwhile, both mutants of ∆Cgsat4 and ∆Cgsat4 exhibited a reduced virulence on host plants [20,24].Deletion of the phosphatase gene CgPPZ1 resulted in a significantly higher potassium content in mycelia [19].The ∆Cgppz1 mutant also attenuated pathogenicity caused by its defects in scavenging reactive oxygen species (ROS) and cell wall integrity [19].These results suggest that potassium homeostasis might be involved in plant infection with phytopathogens.In our previous study, the protein kinase CgSat4 has been involved in potassium uptake and the localization of CgTrk1 [20].However, the roles of Trk proteins regulating potassium uptake and pathogenicity in C. gloeosporioiddes are not clear.
In this study, the potassium uptake transporters Trk1 and Trk2 were identified and characterized in C. gloeosporiodes.Despite high sequence identity between CgTrk1 and CgTrk2, it is likely that CgTrk1 plays dominant roles in potassium homeostasis, invasive growth, and pathogenicity in C. gloeosporiodes.

Fungal Strains and Cultural Conditions
C. gloeosporioides strain SMCG1#C was used as the wild-type (WT) strain for generating gene deletion mutants in the present study [19].All strains were grown on complete medium (CM) agar plates at 28 • C. Mycelia of tested fungal strains were cultured on the liquid CM medium and collected for DNA and RNA extraction as previously described [20].

Generation of Gene-Knockout and Complementary Strains
A polyethylene glycol (PEG)-mediated protoplast transformation method was employed to generate the gene deletion mutants of CgTRK1 and CgTRK2 using a standard one-step gene replacement strategy [24].In brief, about 1.5 kb upstream and downstream flanking regions of the CgTRK1 and CgTRK2 genes were respectively amplified and then fused to the 5 ′ and 3 ′ termini of the hygromycin B phosphotransferase gene (HPH) by PCR, respectively.The PCR products were transformed into protoplasts of the WT as previously described [20].The candidate transformants were screened by PCR analysis and confirmed by Southern blotting [20].
For complementation, a DNA fragment containing the entire open reading frame and its native promoter (CgTRK1 and CgTRK2) was amplified and inserted into the vector pYF11 using the yeast gap-repair approach [29].The resulting complementation constructs pYF11::CgTRK1-GFP and pYF11::CgTRK2-GFP were transformed into protoplasts of the ∆Cgtrk1 and ∆Cgtrk2 mutants to generate the corresponding complemented transformants, respectively.

Determination of Potassium Content in Fungal Mycelia
Mycelial plugs of WT, ∆CgTrk1 and ∆CgTrk2 mutants, respectively, were cultured into liquid CM medium for 2 days.Mycelia were harvested and then washed with ddH 2 O three times.Subsequently, the mycelia were dried using a freeze-dryer and digested with H 2 SO 4 .Then, H 2 O 2 was added to decolorize the prepared samples.Potassium of mycelia Forests 2024, 15, 1044 3 of 11 was measured using a flame spectrophotometer (Perkin Elmer, Waltham, MA, USA) [24].The experiment was conducted three times, and each treatment had three replicates.

Vegetative Growth and Cell Wall Perturbs Resistance
Mycelial plugs of the WT, ∆Cgtrk1 and ∆Cgtrk2 mutants, and the corresponding complemented strains were, respectively, inoculated onto plates of CM, potato dextrose agar (PDA), and minimal medium (MM).These plates were cultured in an incubator at 25 • C [24].At 5 days post-inoculation (dpi), the diameters of the colonies were measured.
To analyze the resistance to cell wall perturbs, mycelial plugs of the tested strains were inoculated onto CM agar plates supplied with calcofluor white (CFW; 100 µg/mL) and Congo red (CR; 200 µg/mL), respectively.Plates were placed in an incubator at 25°C [29].At 5 dpi, the colony diameters were measured.The experiment was conducted three times, and each treatment had three replicates.

Conidiation, Conidial Germination, and Appressorium Formation
For the conidiation assay, the mycelial plugs of WT, mutants, and complemented strains were respectively inoculated into liquid CM medium and shaken at 200 rpm for 24 h at 25 • C. Conidia were harvested by filtering through two layers of Miracloth (EMD Millipore, Bellerica, MA, USA).The filtrate was centrifuged at 8000× g for 5 min.The conidia were resuspended in 1.0 mL of ddH 2 O [28].
Moreover, 20 µL of the conidial suspension (1 × 10 5 conidia/mL) were inoculated onto hydrophobic coverslips at 25 • C. Subsequently, conidial germination and appressorium formation rates were measured at 2 h, 4 h, and 8 h, respectively.To observe invasive hyphae, the conidial suspension (5 × 10 4 conidia/mL) was inoculated onto the onion epidermal layers.The type of invasive hyphae was measured at 18 h post-inoculation.The experiment was conducted with a minimum of 30 measurements per structure under a ZEISS Axio Imager A2m microscope (Carl Zeiss, Göttingen, Germany).

Pathogenicity Assay
For each strain, 10 µL of conidial suspension (1 × 10 5 conidia/mL) containing 0.1% Tween-20 was inoculated onto the healthy leaves of Chinese fir (C.lanceolata), Populus × euramericana cv.Nanlin895, and Liriodendron chinense × tulipifera, respectively.The inoculated leaves were kept in a chamber maintained at 25 • C with 90% humidity and in the dark for the first 24 h, followed by exposure to a light-dark cycle (16-hour light and 8-hour dark cycles).The disease severity was assessed by measuring the length of lesions [19].The experiment was conducted three times, and each treatment had at least three replicates.

Statistical Analyses
Significance analyses were performed with a one-way ANOVA followed by the Duncan's multiple-range test using the SPSS software (SPSS 19.0, IBM, Armonk, NY, USA).Pairwise comparisons were performed using the Student's t-test in Microsoft Excel 2016 version.

Identification and Localization Patterns of CgTrk1
Using the S. cerevisiae Trk1 and Trk2 sequences as the references for searching the genome database of C. gloeosporioides, respectively, we identified two homologous potassium transporter proteins, i.e., CgTrk1 and CgTrk2.Phylogenetic tree analysis showed that Trk1 and Trk2 were highly conserved among phytopathogenic fungi, including C. tropicale, C. siamense, C. higginsianum, Pyricularia oryzae, Fusarium graminearum, and F. oxysporum (Figure 1A).Although the amino acid sequence of CgTrk2 was shorter than CgTrk1, the remaining sequences showed a high identity (Figure S1).
genome database of C. gloeosporioides, respectively, we identified two homologous potassium transporter proteins, i.e., CgTrk1 and CgTrk2.Phylogenetic tree analysis showed that Trk1 and Trk2 were highly conserved among phytopathogenic fungi, including C. tropicale, C. siamense, C. higginsianum, Pyricularia oryzae, Fusarium graminearum, and F. oxysporum (Figure 1A).Although the amino acid sequence of CgTrk2 was shorter than CgTrk1, the remaining sequences showed a high identity (Figure S1).To test the expression and localization patterns of the CgTrk1 protein, the fusion protein of CgTrk1::GFP was individually expressed in the ΔCgtrk1 mutant to generate the complemental strains.Fluorescence microscopic observation showed that CgTrk1::GFP was localized on the plasma membrane in C. gloeosporioides (Figure 1B).
We obtained the gene deletion mutants of CgTRK1 and CgTRK2 using the standard one-step gene replacement strategy in C. gloeosporioides (Figure 1C,D).Candidate mutants of ΔCgtrk1 and ΔCgtrk2 were confirmed by Southern blot, respectively (Figure 1C,D).To test the expression and localization patterns of the CgTrk1 protein, the fusion protein of CgTrk1::GFP was individually expressed in the ∆Cgtrk1 mutant to generate the complemental strains.Fluorescence microscopic observation showed that CgTrk1::GFP was localized on the plasma membrane in C. gloeosporioides (Figure 1B).

Involvement of CgTrk1 in Potassium Uptake and Cell Wall Integrity
We obtained the gene deletion mutants of CgTRK1 and CgTRK2 using the standard one-step gene replacement strategy in C. gloeosporioides (Figure 1C,D).Candidate mutants of ∆Cgtrk1 and ∆Cgtrk2 were confirmed by Southern blot, respectively (Figure 1C,D).

Involvement of CgTrk1 in Potassium Uptake and Cell Wall Integrity
Sequence identities of CgTrk1 and CgTrk2 suggests potential functional similarities.In comparison to the WT, the mycelial potassium content of the ∆Cgtrk1 mutant was significantly reduced.However, the potassium content of the ∆Cgtrk2 mutant was almost similar to that of the WT (Figure 2A).Moreover, we detected the potassium-related genes CgSAT4 (positive factor) and CgPPZ1 (negative factor) in wild-type strains and mutants.Compared to the wild-type strain, the expressional level of CgSAT4 was upregulated in the ∆Cgtrk1 mutant, while the expressional levels of CgPPZ1 were reduced in the ∆Cgtrk1 mutant (Figure 2C).However, the expression of CgSAT4 and CgPPZ1 exhibited no obvious CgTrk1 shares a different role in potassium uptake in C. gloeosporioides.
Mycelial plugs of the tested strains were inoculated onto CM plates with the addition of cell wall-perturbing stressors CFW and CR.The data showed that inhibition of the WT reached ~20% and 25% for CFW and CR, respectively (Figure 2B).However, all mutants of ΔCgtrk1 and ΔCgtrk2 exhibited more resistance to cell wall-perturbing stressors (Figure 2B).Subsequently, we investigated the expression levels of chitin synthases (CHSs) in the mutants of ΔCgtrk1 and ΔCgtrk2, which were attenuated related to wild-type (Figure 2D).These results indicate that CgTrk1 and CgTrk2 are involved in the response to cell wall perturbs.Mycelial plugs of the tested strains were inoculated onto CM plates with the addition of cell wall-perturbing stressors CFW and CR.The data showed that inhibition of the WT reached ~20% and 25% for CFW and CR, respectively (Figure 2B).However, all mutants of ∆Cgtrk1 and ∆Cgtrk2 exhibited more resistance to cell wall-perturbing stressors (Figure 2B).Subsequently, we investigated the expression levels of chitin synthases (CHSs) in the mutants of ∆Cgtrk1 and ∆Cgtrk2, which were attenuated related to wild-type (Figure 2D).These results indicate that CgTrk1 and CgTrk2 are involved in the response to cell wall perturbs.

CgTrk1 Involved in Conidiation and Appressorium Formation
The colony diameter of the ∆Cgtrk1 mutant was smaller than that of the WT and complemented stains on various media plates, while the growth of the ∆Cgtrk2 mutant showed a similar trend to the WT and complemented strain (Figure 3A).The conidiation of ∆Cgtrk1 mutants was significantly decreased compared to the WT and complemented strains.However, the conidiation ability of the ∆Cgtrk2 mutant was similar to that of the WT (Figure 3B).The results indicate that CgTrk1 may be involved in vegetative growth and conidiation in C. gloeosporiodes.During infection, the conidia of C. gloeosporiodes germinate to generate germ tubes and then develop appressoria to penetrate plant cells.Compared to the WT, the conidial germination ratio of ΔCgtrk1 mutant was significantly reduced (Figure 3C).However, there was no difference in conidial germination between the WT and ΔCgtrk2 mutants (Figure 3C).Interestingly, appressorium formation was significantly decreased in the mutants of ΔCgtrk1 and ΔCgtrk2 (Figure 3D).

CgTrk1 Required for Full Virulence
At 5 dpi, all tested strains caused necrotic lesions on the leaves of Chinese fir.compared to the WT and complemented strains.Moreover, lesions caused by the ΔCgtrk1 mutant were significantly smaller.However, there was no difference in the virulence between the WT and ΔCgtrk2 mutants (Figure 4A,B).We also evaluated the pathogenicity of these strains on Populus × euramericana cv.Nanlin895 and Liriodendron chinense × tulipifera.The During infection, the conidia of C. gloeosporiodes germinate to generate germ tubes and then develop appressoria to penetrate plant cells.Compared to the WT, the conidial germination ratio of ∆Cgtrk1 mutant was significantly reduced (Figure 3C).However, there was no difference in conidial germination between the WT and ∆Cgtrk2 mutants (Figure 3C).Interestingly, appressorium formation was significantly decreased in the mutants of ∆Cgtrk1 and ∆Cgtrk2 (Figure 3D).

CgTrk1 Required for Full Virulence
At 5 dpi, all tested strains caused necrotic lesions on the leaves of Chinese fir.compared to the WT and complemented strains.Moreover, lesions caused by the ∆Cgtrk1 mutant were significantly smaller.However, there was no difference in the virulence between the WT and ∆Cgtrk2 mutants (Figure 4A,B).We also evaluated the pathogenicity of these strains on Populus × euramericana cv.Nanlin895 and Liriodendron chinense × tulipifera.The gathered data exhibited the fact that the ∆Cgtrk1 mutant had a decreased virulence compared to the WT, while deletion of CgTRK2 did not show a significant effect on the pathogenicity of C. gloeosporioides (Figure 4C-J).The onion epidermis penetration assays showed that most invasive hyphae of the WT belong to type III and type IV.In the ΔCgtrk2 mutant, the proportion of invasive hyphae of type III and type IV was more than 50%.However, the ΔCgtrk1 mutant had a higher The onion epidermis penetration assays showed that most invasive hyphae of the WT belong to type III and type IV.In the ∆Cgtrk2 mutant, the proportion of invasive hyphae of type III and type IV was more than 50%.However, the ∆Cgtrk1 mutant had a higher proportion of invasive hyphae of type I and type II, reaching 39% and 21%, respectively (Figure 5).proportion of invasive hyphae of type Ⅰ and type Ⅱ, reaching 39% and 21%, respectively (Figure 5).

Discussion
In this study, we identified two potassium transporters in C. gloeosporioides, i.e., CgTrk1 and CgTrk2, which are homologous to ScTrk1 and ScTrk2 in S. cerevisiae, respectively.Microscopic observation has shown that CgTrk1 is distributed along the cell membrane.The potassium content in mycelia was significantly decreased in the mutant ΔCgtrk1 compared to that of the WT.Additionally, it was discovered that CgTrk1, rather than the paralogous protein CgTrk2, plays a dominant role in potassium uptake, conidiation, invasive growth, and pathogenicity in C. gloeosporioides.
Potassium is required for organisms as an essential mineral nutrient, which plays a variety of important roles [3,4,30,31].As potassium is a key monovalent cation, organisms have evolved conserved ion transporters for potassium absorption [4,20].In this study, we identified two high-affinity potassium transporter proteins, i.e., CgTrk1 and CgTrk2, which seem conserved in phytopathogenic fungi.Sequence analyses revealed that there is a high similarity between CgTrk1 and CgTrk2.Our data showed that CgTrk1 was required for potassium uptake; however, the deletion of CgTRK2 has not shown a significant effect on potassium uptake.Additionally, we also observed the expression levels of attenuated CgSat4 , while expression levels of CgPpz1 (the negatively regulated activity of Trk1) were increased in ΔCgrtrk1 mutants.
Fungal cell wall integrity is crucial for normal morphological development and cell survival in hostile environments and plant infection [29].In this study, when the mutants of ΔCgtrk1 and ΔCgtrk2 were exposed to cell wall stressors CFW and CR, they showed similar, stronger resistance compared to the WT.Moreover, expressional levels of CHS(s) in the mutants of ΔCgtrk1 and ΔCgtrk2 exhibited significant attenuation compared to the WT.These data showed the similar function of CgTrk1 and CgTrk2 in regulating cell wall integrity in C. gloeosporioies.Thus, we suggest that CgTrk1 may regulate expressional levels of genes CHS, which are involved in the response to cell wall stressores and phytopathogen infection.
Potassium can to directly and/or indirectly modulate the virulence gene expression, antimicrobial resistance, and biofilm formation in bacteria [10,27,28,32].In our study, deletion of CgTRK1 significantly reduced the vegetative growth, conidiation, appressorium development, invasive hyphae development, and pathogenicity.However, the ΔCgtrk2 mutant just showed a defect in appressorium development.These data indicate the dominant role of CgTrk1 in governing the vegetative growth and pathogenesis in C. gloeosporioides.

Discussion
In this study, we identified two potassium transporters in C. gloeosporioides, i.e., CgTrk1 and CgTrk2, which are homologous to ScTrk1 and ScTrk2 in S. cerevisiae, respectively.Microscopic observation has shown that CgTrk1 is distributed along the cell membrane.The potassium content in mycelia was significantly decreased in the mutant ∆Cgtrk1 compared to that of the WT.Additionally, it was discovered that CgTrk1, rather than the paralogous protein CgTrk2, plays a dominant role in potassium uptake, conidiation, invasive growth, and pathogenicity in C. gloeosporioides.
Potassium is required for organisms as an essential mineral nutrient, which plays a variety of important roles [3,4,30,31].As potassium is a key monovalent cation, organisms have evolved conserved ion transporters for potassium absorption [4,20].In this study, we identified two high-affinity potassium transporter proteins, i.e., CgTrk1 and CgTrk2, which seem conserved in phytopathogenic fungi.Sequence analyses revealed that there is a high similarity between CgTrk1 and CgTrk2.Our data showed that CgTrk1 was required for potassium uptake; however, the deletion of CgTRK2 has not shown a significant effect on potassium uptake.Additionally, we also observed the expression levels of attenuated CgSat4, while expression levels of CgPpz1 (the negatively regulated activity of Trk1) were increased in ∆Cgrtrk1 mutants.
Fungal cell wall integrity is crucial for normal morphological development and cell survival in hostile environments and plant infection [29].In this study, when the mutants of ∆Cgtrk1 and ∆Cgtrk2 were exposed to cell wall stressors CFW and CR, they showed similar, stronger resistance compared to the WT.Moreover, expressional levels of CHS(s) in the mutants of ∆Cgtrk1 and ∆Cgtrk2 exhibited significant attenuation compared to the WT.These data showed the similar function of CgTrk1 and CgTrk2 in regulating cell wall integrity in C. gloeosporioies.Thus, we suggest that CgTrk1 may regulate expressional levels of genes CHS, which are involved in the response to cell wall stressores and phytopathogen infection.
Potassium can to directly and/or indirectly modulate the virulence gene expression, antimicrobial resistance, and biofilm formation in bacteria [10,27,28,32].In our study, deletion of CgTRK1 significantly reduced the vegetative growth, conidiation, appressorium development, invasive hyphae development, and pathogenicity.However, the ∆Cgtrk2 mutant just showed a defect in appressorium development.These data indicate the dominant role of CgTrk1 in governing the vegetative growth and pathogenesis in C. gloeosporioides.
Potassium transporter Trk1 has been identified as a key regulator for facilitating cells to survive in low potassium conditions [9].The Trk1 has been shown to increase potassium influx and decrease the membrane potential, which is beneficial for the reduction of toxic cation accumulation and improvement of salt tolerance [13].In C. gloeosporioides, the phosphatase Ppz1 negatively regulates potassium content and is involved in cell wall integrity, invasive growth, host defense responses, and virulence [16,18].The kinase CgSat4 positively regulates potassium uptake by regulating the subcellular location of potassium transporter CgTrk1 [20].Deletion of CgSAT4 results in abnormal localization of Trk1 in the vacuole, a significant decrease in potassium uptake, and reduced resistance to ion stress [21].However, there is no direct interaction between CgTrk1 and CgSat4.CgSat4 is not involved in the transcription expression of CgTrk1.These data indicate that CgSat4 regulates potassium uptake by regulating the appropriate localization of CgTrk1.Despite the advance, further study is needed on the molecular regulation network of CgTrk1 in the future.

Conclusions
In summary, Trk family proteins facilitate potassium uptake to directly or indirectly govern pathogenicity, development of appressorium and invasive hyphae, and vegetative growth in C. gloeosporioides (Figure 6).
Potassium transporter Trk1 has been identified as a key regulator for facilitating cells to survive in low potassium conditions [9].The Trk1 has been shown to increase potassium influx and decrease the membrane potential, which is beneficial for the reduction of toxic cation accumulation and improvement of salt tolerance [13].In C. gloeosporioides, the phosphatase Ppz1 negatively regulates potassium content and is involved in cell wall integrity, invasive growth, host defense responses, and virulence [16,18].The kinase CgSat4 positively regulates potassium uptake by regulating the subcellular location of potassium transporter CgTrk1 [20].Deletion of CgSAT4 results in abnormal localization of Trk1 in the vacuole, a significant decrease in potassium uptake, and reduced resistance to ion stress [21].However, there is no direct interaction between CgTrk1 and CgSat4.CgSat4 is not involved in the transcription expression of CgTrk1.These data indicate that CgSat4 regulates potassium uptake by regulating the appropriate localization of CgTrk1.Despite the advance, further study is needed on the molecular regulation network of CgTrk1 in the future.

Conclusions
In summary, Trk family proteins facilitate potassium uptake to directly or indirectly govern pathogenicity, development of appressorium and invasive hyphae, and vegetative growth in C. gloeosporioides (Figure 6).

Figure 1 .
Figure 1.Phylogenetic tree of the fungal Trk family and Southern blot analysis of the gene TRK1/2 deletion mutants.(A) The neighbor-joining phylogenetic tree created by using MEGA v7.0.The homologous protein sequences of the Trk family in other phytopathogenic fungi downloaded from the website NCBI (https://www.ncbi.nlm.nih.gov/(24 December 2023)).CsTrk2, CtTrk2, ChTrk2, PoTrk2, FoTrk2, FgTrk2, CsTrk1, CtTrk1, ChTrk1, FgTrk1, FoTrk1, and PoTrk1 from C. tropicale, C. siamense, C. higginsianum, Pyricularia oryzae, Fusarium graminearum, and F. oxysporum, respectively.Green and yellow boxes represent the phylogenetic trees of CgTrk1 and CgTrk2.The red asterisks indicate CgTrk1 and CgTrk2.(B) The distribution of CgTrk1 in the plasma membrane.The fusion protein of CgTrk1::GFP was expressed in hyphae.The scale bar represents 10 µm.(C,D) Southern blot analysis of the genes TRK1/2 deletion mutants.Genomic DNA isolated from the wild-type (WT) strains ΔCgtrk1 and ΔCgtrk2 mutants was digested with restriction enzymes.After being separated with agar gel electrophoresis, the digested DNA was blotted on the membrane.The membrane was hybridized with probes for the ΔCgtrk1 mutant and the probes CgTRK2 and HPH for the ΔCgtrk2 mutant, respectively.The molecular markers are shown in base pairs (bp) on the left.

Figure 1 .
Figure 1.Phylogenetic tree of the fungal Trk family and Southern blot analysis of the gene TRK1/2 deletion mutants.(A) The neighbor-joining phylogenetic tree created by using MEGA v7.0.The homologous protein sequences of the Trk family in other phytopathogenic fungi downloaded from the website NCBI (https://www.ncbi.nlm.nih.gov/(24 December 2023)).CsTrk2, CtTrk2, ChTrk2, PoTrk2, FoTrk2, FgTrk2, CsTrk1, CtTrk1, ChTrk1, FgTrk1, FoTrk1, and PoTrk1 from C. tropicale, C. siamense, C. higginsianum, Pyricularia oryzae, Fusarium graminearum, and F. oxysporum, respectively.Green and yellow boxes represent the phylogenetic trees of CgTrk1 and CgTrk2.The red asterisks indicate CgTrk1 and CgTrk2.(B) The distribution of CgTrk1 in the plasma membrane.The fusion protein of CgTrk1::GFP was expressed in hyphae.The scale bar represents 10 µm.(C,D)Southern blot analysis of the genes TRK1/2 deletion mutants.Genomic DNA isolated from the wild-type (WT) strains ∆Cgtrk1 and ∆Cgtrk2 mutants was digested with restriction enzymes.After being separated with agar gel electrophoresis, the digested DNA was blotted on the membrane.The membrane was hybridized with probes for the ∆Cgtrk1 mutant and the probes CgTRK2 and HPH for the ∆Cgtrk2 mutant, respectively.The molecular markers are shown in base pairs (bp) on the left.

Figure 2 .
Figure 2. The potassium content was measured in the mycelia and cell wall perturbing stress resistance for ΔCgtrk1 and ΔCgtrk2 mutants.(A) The fungal mycelia were harvested from liquid CM medium after cultured for 2 days.Mycelia was dried using a freeze dryer and digested with 98% H2SO4.Moreover, it was restored to colorless in order to prepare the mycelial digestion solution.The digestions were measured using a flame spectrophotometer.The unit "µg/mg" represents "potassium/mycelium". (B) The fungal mycelia blocks of SMCG1#C, ΔCgtrk1, and ΔCgtrk2 mutants and complemented strains were inoculated onto CM medium with 100 µg/mL CFW and 200 µg/mL CR.The inhibition rate of mycelia growth was measured at inoculated five days.CFW: Calcofluor white; CR: Congo red.(C,D) The expressional levels of potassium-related genes (C) and chitin sythases (D) were significantly attenuated in mutants.Total RNA was extracted from fungal mycelia balls of the SMCG1#C, ΔCgtrk1 and ΔCgtrk2 mutants.The expressional levels of potassium-related genes (C) and CHSs (D) were detected by RT-qPCR, with the 18S ribosomal RNA (18S rRNA) as a reference gene.Error bars represent the standard deviation.Asterisks indicate a significant difference at p < 0.01.

Figure 2 .
Figure 2. The potassium content was measured in the mycelia and cell wall perturbing stress resistance for ∆Cgtrk1 and ∆Cgtrk2 mutants.(A) The fungal mycelia were harvested from liquid CM medium after cultured for 2 days.Mycelia was dried using a freeze dryer and digested with 98% H 2 SO 4 .Moreover, it was restored to colorless in order to prepare the mycelial digestion solution.The digestions were measured using a flame spectrophotometer.The unit "µg/mg" represents "potassium/mycelium". (B) The fungal mycelia blocks of SMCG1#C, ∆Cgtrk1, and ∆Cgtrk2 mutants and complemented strains were inoculated onto CM medium with 100 µg/mL CFW and 200 µg/mL CR.The inhibition rate of mycelia growth was measured at inoculated five days.CFW: Calcofluor white; CR: Congo red.(C,D) The expressional levels of potassium-related genes (C) and chitin sythases (D) were significantly attenuated in mutants.Total RNA was extracted from fungal mycelia balls of the SMCG1#C, ∆Cgtrk1 and ∆Cgtrk2 mutants.The expressional levels of potassium-related genes (C) and CHSs (D) were detected by RT-qPCR, with the 18S ribosomal RNA (18S rRNA) as a reference gene.Error bars represent the standard deviation.Asterisks indicate a significant difference at p < 0.01.

Figure 3 .
Figure 3. Trk family proteins involved in vegetable growth, conidiation, conidial germination, and appressorium formation.(A) The colony diameter of SMCG1#C, ΔCgtrk1, and ΔCgtrk2 mutants, and the complemented strain grown on CM, PDA, and MM plates for 5 days.(B) The conidiation of SMCG1#C, ΔCgtrk1, and ΔCgtrk2 mutants and complemented strains cultured in liquid CM.(C,D) Conidial germination (C) and appressorium formation (D) rates of SMCG1#C, ΔCgtrk1, and ΔCgtrk2 mutants and complemented strains were calculated after being inoculation onto hydrophobic cover slips for 2 h, 4 h, and 8 h, respectively.Error bars represent the standard deviation.Asterisks indicate a significant difference at p < 0.01.

Figure 3 .
Figure 3. Trk family proteins involved in vegetable growth, conidiation, conidial germination, and appressorium formation.(A) The colony diameter of SMCG1#C, ∆Cgtrk1, and ∆Cgtrk2 mutants, and the complemented strain grown on CM, PDA, and MM plates for 5 days.(B) The conidiation of SMCG1#C, ∆Cgtrk1, and ∆Cgtrk2 mutants and complemented strains cultured in liquid CM.(C,D) Conidial germination (C) and appressorium formation (D) rates of SMCG1#C, ∆Cgtrk1, and ∆Cgtrk2 mutants and complemented strains were calculated after being inoculation onto hydrophobic cover slips for 2 h, 4 h, and 8 h, respectively.Error bars represent the standard deviation.Asterisks indicate a significant difference at p < 0.01.

ForestsForests
2024,15,  x FOR PEER REVIEW 7 of 11 gathered data exhibited the fact that the ΔCgtrk1 mutant had a decreased virulence compared to the WT, while deletion of CgTRK2 did not show a significant effect on the pathogenicity of C. gloeosporioides (Figure4C-J).

Figure 4 .
Figure 4. CgTrk1 required for full virulence in C. gloeosporioides.(A,B) Ten µL of conidial suspensions of SMCG1#C, mutants of ΔCgtrk1 and ΔCgtrk2, and complemented strains were inoculated onto the leaves of Chinese fir in a moist chamber at 25 °C with high humidity.Pictorial (A) and quantitative (B) representations of lesion length were obtained 5 dpi.(C-F) Pathogenicity assays of ΔCgtrk1 and ΔCgtrk2 mutants on leaves of L. dronchinensis × tulipifera, and Populus × euramericana cv.Nanlin895 for 5 days.Upper: Representative disease symptoms of the WT, mutants, and complemented strains (C-F).Down: Diameter of lesion measured at 5 dpi (G-J).Scale bars indicate 10 mm.Error bars represent the standard deviation.Asterisks indicate a significant difference at p < 0.01.

Figure 4 .
Figure 4. CgTrk1 required for full virulence in C. gloeosporioides.(A,B) Ten µL of conidial suspensions of SMCG1#C, mutants of ∆Cgtrk1 and ∆Cgtrk2, and complemented strains were inoculated onto the leaves of Chinese fir in a moist chamber at 25 • C with high humidity.Pictorial (A) and quantitative (B) representations of lesion length were obtained 5 dpi.(C-F) Pathogenicity assays of ∆Cgtrk1 and ∆Cgtrk2 mutants on leaves of L. dronchinensis × tulipifera, and Populus × euramericana cv.Nanlin895 for 5 days.Upper: Representative disease symptoms of the WT, mutants, and complemented strains (C-F).Down: Diameter of lesion measured at 5 dpi (G-J).Scale bars indicate 10 mm.Error bars represent the standard deviation.Asterisks indicate a significant difference at p < 0.01.

Figure 5 .
Figure 5.The conidial suspension was inoculated onto the adaxial surface of onion epidermal.At 18 h post-inoculation, IH was observed under a microscope.Four types of IH are as follows: type I, no hyphae penetration; type II, IH with one branch; type III, IH with at least two branches but having limited expansion; and type IV, IH with numerous branching and extensive hyphal growth.

Figure 5 .
Figure 5.The conidial suspension was inoculated onto the adaxial surface of onion epidermal.At 18 h post-inoculation, IH was observed under a microscope.Four types of IH are as follows: type I, no hyphae penetration; type II, IH with one branch; type III, IH with at least two branches but having limited expansion; and type IV, IH with numerous branching and extensive hyphal growth.

Figure 6 .
Figure 6.A proposed model elucidating the involvement of potassium homeostasis-related proteins (Trk1, Sat4, and Ppz1) in C. gloeosporioides.In essence, intracellular potassium homeostasis relies on the high-affinity ion transporter CgTrk1, which governs a plethora of fundamental life activities, such as ion homeostasis, cell wall integrity, pathogenicity, and response to stresses in C. gloeosporioides.Under conditions of potassium deficiency, the activated Ser/Thr kinase Sat4 phosphorylates the potassium transporter Trk1 and subsequently regulates the subcellular localization of Trk1 to the plasma membrane for potassium uptake from the extracellular space.Conversely, in potassiumrich environments, the phosphatase Ppz1 inhibits Trk1 activity through dephosphorylation, thereby preventing the function of the potassium transporter CgTrk1 in response to ion toxicity.