A major facilitator superfamily transporter PlMFS1 contributes to growth, oosporogenesis, and pathogenesis of Peronophythora litchii

Abstract

Major facilitator superfamily (MFS) transporters are secondary active membrane transporters that play an important role in solute interchange and energy metabolism. Peronophythora litchii causes the most destructive disease on lichi, litchi downy blight. PIM90 was reported as a key oosporogenesis regulator. Here, we identified an MFS transporter gene PIMFS1, which is up-regulated during oospore formation at the late infection stage, while down-regulated in the PIM90 mutant. To investigate PIMFS1 function, we generated PIMFS1 knockout mutants using CRISPR/Cas9-mediated genome editing technology. Compared with the wild-type strain SHS3, PIMFS1 deletion impaired mycelium growth, zoospore release, oospore production and pathogenicity. Furthermore, PIMFS1 deletion significantly affected P. litchii utilization of fructose, lactose and maltose, and may be the PIMFS1 mechanism involved in mycelial growth. PIMFS1 gene deletion also led to deceased laccase activity, laccase-encoding gene downregulation and impaired P. litchii pathogenicity. To our knowledge, this is the first report of an MFS transporter involved in sugar utilization, sexual reproduction, asexual reproduction and pathogenesis in oomycetes.

Headnote

Received 13 April 2023; Accepted 7 September 2023; Available online 21 December 2023

ABSTRACT

Major facilitator superfamily (MFS) transporters are secondary active membrane transporters that play an important role in solute interchange and energy metabolism. Peronophythora litchii causes the most destructive disease on lichi, litchi downy blight. PIM90 was reported as a key oosporogenesis regulator. Here, we identified an MFS transporter gene PIMFS1, which is up-regulated during oospore formation at the late infection stage, while down-regulated in the PIM90 mutant. To investigate PIMFS1 function, we generated PIMFS1 knockout mutants using CRISPR/Cas9-mediated genome editing technology. Compared with the wild-type strain SHS3, PIMFS1 deletion impaired mycelium growth, zoospore release, oospore production and pathogenicity. Furthermore, PIMFS1 deletion significantly affected P. litchii utilization of fructose, lactose and maltose, and may be the PIMFS1 mechanism involved in mycelial growth. PIMFS1 gene deletion also led to deceased laccase activity, laccase-encoding gene downregulation and impaired P. litchii pathogenicity. To our knowledge, this is the first report of an MFS transporter involved in sugar utilization, sexual reproduction, asexual reproduction and pathogenesis in oomycetes.

Keywords: Peronophythora litchii; Major facilitator superfamily; Oospore; Pathogenicity

1. Introduction

Oomycetes are fungus-like organisms in the Stramenopiles that are evolutionarily close to diatoms and brown algae, but phylogenetically distant from true fungi (Tyler et al., 2006; Cavalier-Smith et al., 2018). Oomycetes include several plant pathogens that can severely damage a wide range of plants and threaten agricultural production and ecosystems (Kamoun et al., 2015). For example, it was estimated that worldwide vegetable production valued at over one billion dollars is annually threatened by Phytophthora capsici alone (Lamour et al., 2012; Shi et al., 2024). Phytophthora sojae, the causal agent of soybean root and stem rot, causes yield losses of approximately $1-2 billion annually (Tyler, 2007), and Phytophthora infestans, the potato late blight pathogen, led to the Irish potato famine in the 19th century (Birch and Whisson, 2001).

Litchi (Litchi chinensis Sonn.) is a highly valued tropical and subtropical fruit due to its appearance, flavor and nutritional qualities (Jiang et al., 2006; Wang et al., 2023). Litchi downy blight caused by Peronophythora litchii is one of the most destructive diseases in litchi production and storage. However, few studies have been conducted on P. litchii gene functions, hence more research is required to reveal the mechanism of its growth, development and pathogenesis (Jiang et al., 2018; Kong et al., 2019; Huang et al, 2021; Wang et al, 2022; Zhu et al., 2022). With the completion of P. litchii genome and transcriptome sequencing, as well as the recent development of CRISPR/Cas9 genome editing technology (Ye et al., 2016; Sun et al., 2017; Situ et al., 2020; Wang et al., 2024), investigation on P. litchii development and pathogenesis at the molecular level has been accelerated and will provide a novel theoretical foundation for creating new disease control strategies.

The major facilitator superfamily (MFS) is one of the two largest membrane transporter superfamilies in eukaryotes and prokaryotes (Liu et al., 2017). MES can transport small molecules by chemiosmotic ion gradients or electrochemical proton motive force and usually consists of 12 or 14 transmembrane segments (TMSs) with intervening intracellular and extracellular loops (Pao et al., 1998). According to the current transporter classification database (TCDB, www.tcdb.org, 2021), the Conidiation and Conidial Germination Protein (CCGP), Organo Anion Transporter (OAT), and Ferroportin (Fpn) families, and other subfamilies are among the 15 subfamilies that make up the main facilitator superfamily. Its substrate diversity endows the MFS superfamily with a significant role in solute interchange and energy metabolism (Sun et al., 2011). Additionally, MFS transporters perform various functions in different organisms. In Arabidopsis, zincinduced facilitator 1 (ZIF1), has been described as a tonoplastlocalized transporter involved in zinc tolerance (Haydon and Cobbett, 2007), indicating that MFS transporters influence plant ion homeostasis. In plant pathogenic fungi, MFS transporters can pump fungal toxins that contribute to increased fungal aggressiveness to host plants (de Ramón-Carbonell et al, 2019). Furthermore, overexpression of MFS transporters could confer resistance to a wide range of chemical drugs simultaneously, thus conferring the fungus a multidrug resistance (MDR) phenotype (de Ramón-Carbonell et al, 2019). A Botrytis cinerea MES transporter is not only involved in isothiocyanates (ITCs)-efflux, but also a virulence factor providing tolerance to glucosinolatebreakdown products (Vela-Corcia et al., 2019). In Colletotrichum gloeosporioides, CgMFS1, is required for sugar transport, oxidative stress resistance, and pathogenicity (Liu et al., 2021). AaMFS1 is an efflux pump for TeA transmembrane transport and pathogenicity in Alternaria alternata (Sun et al., 2022). Colletotrichum fructicola CfMfs1 plays a role in conidiation, sugar transport, stress response, conidial penetration, appressorial turgor pressure and virulence in tea oil camellia (Chen et al., 2019).

We identified an MFS transporter gene, PIMFS1, which is down-regulated in the PIM90 mutant reported previously (Jiang et al, 2017), and found that it and its orthologs are conserved in oomycetes, and up-regulated during oospore formation and plant infection. PIMFS1 deletion impaired pathogen growth, likely due to defects in sugar utilization. Meanwhile, PIMSF1 is also involved in P. litchii oospore formation, laccase activity and pathogenicity. This study will provide new insight into the MFS transporter mechanism involved in oomycete growth, sexual reproduction and plant infection.

2. Materials and methods

2.1. Bioinformatics analysis

All MFS-domain protein sequences were obtained from NCBI (https://pubmed.ncbi.nlm.nih.gov) and FUNGIDB (http://fungidb. org/fungidb) and submitted to NCBI-CDD (https://www.ncbi. nlm.nih.gov/cdd/) and SMART (https://smart.embl.de) to identify conserved domains. Sequence alignments were created with the ClustalW program (Thompson et al., 1994) and a phylogenetic tree was constructed with the MEGA-X program with a neighborjoining algorithm using 1 000 bootstrap replicates (Kumar et al, 2018). Protein homology modeling was performed with SWISSMODEL (Waterhouse et al., 2018).

2.2. Microbial strains and culture conditions

The P. litchii strain SHS3 was cultured on carrot juice agar (CJA) medium (juice from 200 в carrot topped up to 1 L, agar 15 g-L7· for solid medium) at 25 °C in the dark. RNA was extracted from vegetative mycelia (MY), sporangia (SP), zoospores (ZO), cysts (CY), germinated cysts (GC) and oospores (00), as well as infection stages (IF 1.5-IF 24: 1.5, 3, 6, 12, and 24 h post-inoculation), as previously described (Ye et al., 2011; Jiang et al., 2017).

For growth rate analysis, 5 mm x 5 mm mycelium plugs were inoculated on CJA medium plates and diameters were measured after 7 days. For mycelium staining, fresh hyphae were stained with calcofluor white for 1 min and then observed under a fluorescent microscope (Ouyang et al., 2019; Shetty et al., 2019). For growth under different sugar sources, 5 mm x 5 mm mycelium plugs were inoculated on plates with Petri-medium using fructose, lactose, sucrose, maltose or glucose as a carbon source. For sensitivity to different stresses, 5 mm x 5 mm mycelium plugs were inoculated on plates with Petri-medium supplemented with 1,2 or 5 mmol-L · H,O»; 0.1, 0.2 or 0.3 mmol-L-· CaCl,; 0.05, 0.1 or 0.2 mol-L7· NaCl or 1 mol-L · Sorbitol (Zhu et al., 2022). Diameters were measured seven days after inoculation and the inhibition rate was calculated from diameter growth. The experiments were repeated three times for each assay in triplicate. The data were subjected to statistical analysis using a two-tailed t-test.

2.3. Nucleic acid manipulation and quantitative PCR assay

SHS3 was grown on CJA medium at 25 °C in the dark. RNA was extracted from mycelia (MY), sporangia (SP), zoospores (ZO), cysts (CY), germinated cysts (GC), oospores (Оо) and infection stages (IF 1.5-IF 24 h) using an All-In-One DNA/RNA Mini-preps Kit (Bio Basic, Toronto, Canada) according to the recommended protocol. All cDNAs were synthesized from total RNA by PrimeScript RT Master Mix (TaKaRa). Quantitative PCR was performed in 20 pL reactions that contained 20 ng cDNA, 0.4 umol-L specific primer of PIMFS1, 10 uL SYBR Premix Ex Taq II (TaKaRa) and 6.4 pL DEPCtreated water. The qPCRs were performed on qTOWER3 real-time PCR thermal cyclers (Analytik Jena, Germany) under the following conditions: 95 °C for 2 min, 40 cycles at 95 °C for 30 5, and 60 °C for 30 s to calculate cycle threshold values, followed by a dissociation program of 95 °C for 15 в, 60 °C for 1 min, and 95 °C for 15 s to obtain melt curves. The Actin gene from P. litchii was used as an internal control and relative expression levels were calculated with the formula 2-44 (Livak and Schmittgen, 2001).

2.4. CRISPR/Cas9-mediated genome editing

All the primers used in this study are listed in Table S1. To generate gene replacement constructs (pBluescript П KS-PIMFS1), 1 kb long upstream/downstream arms of the PIMFS1 coding region were amplified from P. litchii genomic DNA by Phanta Max Super-Fidelity DNA Polymerase (Vazyme, Nanjing, China) using primer pairs PIMFS1-L-F/R, PIMFS1-R-F/R and inserted into pBluescript II KS vector digested by Smal by ClonExpress MultiS II One Step Cloning Kit (Vazyme, Nanjing, China). Potential offtarget sites were examined using the web tool EuPaGDT against the P. litchii genome. Two sgRNAs were linked to the linearized pYF2.3G-Ribo-sgRNA, which were digested by Nhe I and Bsa 1 (New England Biolabs, Massachusetts, U.S.A.), respectively.

To generate PIMFSlknockout transformants, two sgRNA, pBluescript II KS-PIMFS1 and the hSpCas9 expressing plasmid were co-transformed into SHS3 protoplasts using polyethylene glycol (PEG)-mediated protoplast transformation as described previously (Fang and Tyler, 2016; Jiang et al., 2017). The transformed protoplasts were recovered overnight and then selected on CJA medium supplemented with 30 ug:mL · G418. After 2-3 days, the primary transformants were transferred to new selective media separately, and genomic DNA (gDNA) was extracted from them. The mutants were identified by genomic PCR with the primers PIMFS1BW-F and PIMFS1BW-R, confirmed by Sanger sequencing, and used for subsequent sequencing analysis.

2.5. Development of zoospore and oospore assays

For sporangia collection, five 5 mm x 5 mm mycelium plugs from 5-day-old mycelia were placed into 2.5 mL sterile water to prepare a sporangium suspension. Sporangia were detached from the sporangiophore in water and 1 uL of suspension was counted for sporangium production. Then, sporangium suspensions were incubated at 16 °C for 2 h, and 1 pL of suspension was counted for zoospore production. For oospore production, 5 mm x 5 mM Mycelium plugs were cultured on CJA medium at 25 °C in the dark for 21 days. Then, five 5 mm x 5 mm mycelium plugs were homogenized with 0.5 mL sterile water in 7, 14 and 21 days post-inoculation, and 500 pL of suspensions were used for calculation of oospore production. All experiments were performed independently three times. The data were subjected to statistical analysis using a two-tailed t-test.

2.6. Pathogenicity assay

For pathogenicity assay, tender litchi (Guiwei) leaves were collected from the litchi orchard at the South China Agricultural University, Guangzhou, Guangdong province. One hundred zoospores or 5 mm x 5 mm mycelium plugs of each strain were inoculated on the center of the tender leaf, and incubated at 25 °C and 80% humidity in the dark for 12 h. Lesion diameter was measured at 48 hpi (hours post inoculation) and then photos were taken. The experiments were repeated independently three times.

2.7. Laccase activity assays

Laccase activity was analyzed according to a previously described procedure in Li et al. (2021). 5 mm x 5 mm mycelium plugs were inoculated on lima bean agar (LBA) medium supplemented with 0.2 mmol-L7· 2,29-azino-di-3ethylbenzathiazoline-6-sulfonate (ABTS, Sigma-Aldrich, USA) at 25 °C for 20 days in the dark. The experiments were repeated independently three times, and in each experiment, there were three replicates for each strain.

3. Results

3.1. PIMFS1 is down-regulated in M90 mutants and encodes a conserved major facilitator superfamily transporter

M90 is critical for oosporogenesis in oomycetes, such as in P. infestans, P. litchii, and Pythium ultimum (Jiang et al, 2017; Feng et al, 2021). Based on the PIM90-silenced mutant transcriptome database and verified by quantitative RT-PCR experiments, we found a major facilitator superfamily transporter gene, designated PIMFS1, was down-regulated to 1.5% in the transcriptome and 4% of the wild type in PIM90 mutant, respectively (Fig. 1, A). The PIMFS1 gene length was 1431 bp without intron, and encoded a protein of 476-amino acid (aa) containing 12 transmembrane segments (Fig. 1, B and C). PIMFS1 is annotated as a member of the Glycoside-PentosideHexuronide (GPH): Cation Symporter Family, which can uptake sugars in symport with either an H· or Na· cation (Reinders and Ward, 2001). PIMFS1predicted 3D structure is highly similar (0.82 coverage) to the monocarboxylate transporter (SfMCT) of the SLC16 solute carrier family responsible for 1-lactate transport across the plasma membrane in human and other mammalian cells (Bosshart et al., 2019) (Fig. 1, С). Additionally, we searched the orthologous proteins from other oomycetes, fungi and model organisms for sequence alignment and phylogenetic analysis and found that PIMFS1orthologs are widespread and conserved in oomycetes and fungi (Fig. 1, D and Fig. S1).

3.2. PIMFS1 was highly expressed during sexual reproduction and in late infection stage

To investigate PIMFS1 functions in P. litchii growth, development and infection, we analyzed the PIMFS1 gene transcriptional pattern by qRT-PCR with the samples (mycelia, sporangia, zoospores, cysts, germinating cysts, oospores, and infection stages, including 1.5, 3, 6, 12 and 24 hpi). Compared to the mycelia stage, PIMFS1 was up-regulated in the oospore stage and the late stage of plant infection (24 hpi) (Fig. 1, E). These results suggest that PIMFS1 may play an important role in P. litchii oosporogenesis and pathogenesis.

3.3. PIMFS1 knockout mutant generation by CRISPR/Cas9 genome editing technology

Using the CRISPR/Cas9 genome editing technique, we generated PIMFS1 knockout mutants in the P. litchii wild-type strain SHS3. Two single guide RNAs (sgRNA) were designed using the web tool EuPaGDT (http://grna.ctegd.uga.edu/) and used to disrupt the PIMFS1 coding region. We constructed a homologous donor template (pBluescript II KS-PIMFS1) to improve homologydirected repair (HDR) efficiency during genome editing (Fig. 2, A). Two sgRNA expression plasmids, pBluescript II KS-PIMFS1, and the hSpCas9 expression plasmid were co-transformed into wild type strain SHS3 using PEG-mediated protoplast transformation. We successfully generated two mutants, T16 and T34, which were verified by PCR amplification and confirmed by Sanger sequencing (Fig. 2, B and C). A 1 248 bp fragment was deleted between the two sgRNA target sites in the mutants T16 and T34. A transformant that failed to acquire PIMFS1 mutation was selected as the control (CK) strain. These results demonstrated that PIMFS1 was knocked out in T16 and T34.

3.4. By participating in sugar utilization, PIMFS1 is involved in mycelium growth

Next, we analyzed PIMFS1 mutant phenotypes throughout the P. litchii life cycle. Compared to WT and CK, the mutants had a significantly reduced growth rate on carrot juice agar (CJA) medium on the 7th day after inoculation (Fig. 3, A and B). To explore the effect of PIMFS1 on mycelium growth, we stained the hyphae with calcofluor white staining and observed them microscopically. There was no significant difference in mycelium and sporangia morphology between the mutants and WT, indicating that PIMFS1 is required for mycelium growth.

Next, we inoculated the WT and PIMFS1 mutants (T16 and T34) on Plich medium supplemented with fructose, lactose, sucrose, maltose or glucose, to observe their different carbohydrate utilization capacities. After culturing in the dark for 7 days, mutant mycelial growth was significantly restricted when grown on media with an exogenous addition of fructose, lactose or maltose. Conversely, mutant hyphae on glucose or sucrose media were comparable to those of WT (Fig. 3, C and D). These results demonstrate that loss of PIMFS1 significantly compromised the ability of P. litchii to utilize nutrients such as fructose, lactose, and maltose in the medium, resulting in restricted mycelial growth.

3.5. PIMFS1 is associated with P. litchii oosporogenesis and zoospore release

To explore whether PIMFS1 is involved in oospore production, WT, CK and knockout mutants were inoculated on CJA medium, and oospore production was calculated at 7, 14 and 21 days after inoculation. Compared with WT and CK, mutant oospore morphology and size were not significantly different, but PIMFS1 mutant oospore numbers were significantly reduced (Fig. 4), indicating that PIMFS1 is important for oospore production.

3.6. PIMFS1 is required for P. litchii full virulence

PIMFS1 expression level was significantly higher in the latter infection stages, suggesting that it may play a role in P. litchii pathogenicity. We inoculated tender litchi leaves with spore suspension (100 zoospores) (Fig. 5, A and C) or mycelial plugs (Fig. 5, B and D) containing mycelium from the WT, CK or PIMFS1 mutants and observed lesion diameter at 48 hpi. Mutant-infected leaves had significantly smaller lesions than WT or CK, suggesting that PIMFS1 is required for P. litchii pathogenesis.

In the interaction process between pathogenic microorganisms and host plants, laccase secreted by pathogenic microorganisms plays an important role in removing active oxygen accumulation and callose deposition (Kong et al., 2020). PIMFS1 knockout resulted in a significant decrease in P. litchii pathogenicity, so we tested whether the decreased pathogenicity was related to laccase level changes. We inoculated the WT and knockout mutants on Plich medium supplemented with ABTS to detect laccase activity and found that the mutants had no detectable laccase activity even when inoculation was extended to 20 days, while WT and CK strains produced a large area of purple material (Fig. 6) indicating laccase activity. These results revealed that PIMFS1 is critical for virulence, likely via regulating P. litchii laccase activity.

3.7. PIMFS1 is involved in zoospore release and response to sorbitol stress

In fungi, MFS transporter is involved in asexual spore development (Chen et al, 2019). Here, we analyzed PIMFS1 mutant asexual development and its response to various stresses (H,O, SDS, CR, CFW, CaCl,, NaCl and sorbitol). Compared to WT, mutants showed no significant difference in sporangial production, cyst germination or response to H,0, SDS, CR, CFW or CaCl, (Fig. 7, A and C-F, Fig. 52). However, PIMFS1 mutants showed reduced zoospore release rates and were more sensitive to sorbitol (Fig. 7, В and E). These results indicate that PIMFS1 is involved in zoospore release and response to osmotic stress.

4. Discussion

MFS transporter is one of the largest conserved secondary active transporters and is widespread and conserved in animals, plants, fungi and oomycetes (Yan, 2013). Nowadays, increasingly more physiological functions of the MFS transporter have been revealed (Liu et al., 2017; Chen et al., 2019). However, specific MFS functions in oomycetes are still unknown. We identified PIMFS1 in PIM90 mutant transcriptional data and determined that it is associated with P. litchii mycelial growth, oospore yield and pathogenicity. Moreover, it is related to laccase activity and sugar utilization.

In P. litchii asexual development, PIMFS1 knockout did not affect sporangium yield and morphology, but significantly affected zoospore release rates from sporangia. C. fructicola CfMfs1 has a role in conidiation, sugar transport, conidial penetration, and appressorial turgor pressure (Chen et al., 2019). Intrahyphal hyphae formation and conidiation production were impaired in Colletotrichum higginsianum ChMfs1 the knockout mutant which could be fully restored by genetic complementation of the ChMfs1 gene (Liu et al., 2017). Our results showed that PIMFS1 mutants displayed lower utilization of fructose, lactose and maltose. As the in-situ complementation system P. litchii was being established, we did not obtain a PIMFS1 mutant supplementation strain. However, two independent mutants generated using CRISPR/Cas9 technology exhibited the same phenotype in growth and sugar utilization, and that is consistent with the structure prediction suggesting that PIMFS1 may be responsible for sugar transportation. These studies suggest that the fungal and oomycete MFS transporter showed various asexual development functions. Although PIMFS1 is required for sugar utilization, the connection between sugar utilization and P. litchii development remains ambiguous.

The PIMFS1 transcript expression level in the late stage of P. litchii infection reached 100 times that of the hyphae, and the pathogenicity test showed that PIMFS1 deletion significantly reduced the virulence. Through genome sequence analysis, we found that PIMFS1 is not located in the gene cluster of pathogenicrelated functional genes, and the reduced pathogenicity caused by PIMFS1 knockout may be due to the loss of the ability to absorb and transport carbohydrates, resulting in reduced hyphal growth and development. In C. higginsianum ChMfs1 is also important for pathogenicity (Liu et al., 2017). In Cercospora nicotianae, deletion of an MFS transporter-like gene reduced cercosporin toxin accumulation and fungal virulence (Choquer et al., 2007). In C. gloeosporioides, a major facilitator superfamily transporter, CgMFS1 is required for sugar transport, oxidative stress resistance, and pathogenicity (Liu et al., 2021). Notably, in our study PIMFS1 deletion caused a significant decrease in laccase activity (either by production being blocked or its activity decreased), and the pathogenicity attenuated. However, the PIMFS1 molecular mechanism for P. litchii pathogenicity and whether PIMFS1 directly participates in the infection require further research. It appears that the MFS transporter is involved in pathogenicity by different mechanisms in various fungi or oomycetes but the direct relationship between virulence and sugar transport is unclear.

Sexual reproduction is an important part of the P. litchii life cycle (Situ et al., 2022). Oospore presence can help the pathogen to successfully multiply and resist stress, which creates great difficulties for the prevention and control of litchi downy blight. PIM90 is involved in P. litchii sexual and asexual differentiation. The PIM90-silenced transformants displayed a drastically reduced capacity for producing sexual spores and a delay in zoospore release and encystment stages (Jiang et al., 2017). In this study, PIMFS1 deletion also significantly reduced oospore production. Furthermore, in the PIM90 transcriptome database, we found that PIM90 silencing significantly reduced PIMFS1 expression. We speculate that PIM90 may be upstream of PIMFS1 and indirectly regulate it; however, the relationship between PIMFS1 and PIM90 needs further study.

Sexual reproduction is an important feature of the oomycete life cycle, which generates thick-walled sexual spores called 00spores that play key roles in many plant diseases (Grünwald and Flier, 2005). In P. litchii sexual reproduction, PIMFS1 knockout affected oospore yield, and our results suggest that PIMFS1 plays an important role in oospore production. Meanwhile, a C,H, zinc finger protein PICZF1 is also necessary for oospore development (zhu et al, 2022). In another oomycete, P. ultimum, PuFLP targeted by M90 was also involved in sexual reproduction (Feng et al, 2021). We identified a new component in the M90-mediated oospore formation signaling pathway; however, more studies are needed in the future to reveal the regulatory network of oomycete sexual reproduction.

5. Conclusions

We used CRISPR/Cas9-mediated gene editing technology to generate PIMFS1 mutants and explored PIMFS1 functions. PIMFS1 plays a key role in mycelial growth, and in P. litchii, likely via the utilization of fructose, lactose and maltose. PIMSF1 also regulates sexual reproduction, laccase activity and pathogenicity. To our knowledge, this study is the first to reveal a major facilitator superfamily member involved in P. litchii growth, development and pathogenicity.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We thank Professor Yizhen Deng (South China Agricultural University) for editing the article. This work was funded by the Natural Science Foundation of Guangdong Province, China (Grant Nos. 2023A1515012617, 2022A1515010458 and 2023A1515030267), Guangzhou Science & Technology Program (Grant No. 202201010410) and the earmarked fund for CARS-32.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.1016/j.hpj.2023. 09.003.

Sidebar

Peer review under responsibility of Chinese Society of Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy of Agricultural Sciences (CAAS)

Footnote

1 These authors have contributed equally to this work.

* Corresponding author.

E-mail address: [email protected]

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