UvSMEK1, a Suppressor of MEK Null, Regulates Pathogenicity, Conidiation and Conidial Germination in Rice False Smut Fungus Ustilaginoidea virens

Yu Junjie, Yu Mina, Song Tianqiao, Cao Huijuan, Yong Mingli, Pan Xiayan, Qi Zhongqiang,Du Yan, Zhang Rongsheng, Yin Xiaole, Liang Dong, Liu Yongfeng, 2

Research Paper

UvSMEK1, a Suppressor of MEK Null, Regulates Pathogenicity, Conidiation and Conidial Germination in Rice False Smut Fungus

Yu Junjie1, Yu Mina1, Song Tianqiao1, Cao Huijuan1, Yong Mingli1, Pan Xiayan1, Qi Zhongqiang1,Du Yan1, Zhang Rongsheng1, Yin Xiaole1, Liang Dong1, Liu Yongfeng1, 2

(Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China; School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang 212013, China)

Rice false smut, which is caused by, is an emerging disease of rice spikelets in rice-growing areas worldwide. However, the infection mechanism ofon rice spikelets is still unclear. Here, we characterized a suppressor of mitogen-activated protein kinase kinase or ERK kinase (MEK) null () inthat is conserved among filamentous fungi. Compared with wild typestrain P-1,deletion mutants were defective in pathogenicity and conidial germination. In addition, conidiation of UvSMEK1 deletion mutants was significantly reduced on yeast extract tryptone (YT) plates, but increased in YT broth compared with the wild type. Compared withexpression level during the vegetative mycelia and conidiation stages,dramatically increased during infection of rice florets. Surprisingly, thedeletion mutants exhibited higher tolerance to H2O2and NaCl. In summary, presented evidence suggested that UvSMEK1 positively regulated pathogenicity, conidial germination and conidiation in YT broth, and negatively regulated conidiation on YT medium and tolerance to oxidative and osmotic stresses. The results enhance our understanding of the regulatory mechanism of pathogenicity of, and present a potential molecular target for blocking rice infection by.

suppressor; MEK null;; pathogenicity; conidial germination; conidiation

Rice false smut (RFS) has become a devasting disease in China in the recent decades due to the large-scale cultivation of high-yielding cultivars and the use of chemical fertilizers (Sun et al, 2013; Tang et al, 2013; Yu et al, 2019). The causal agentinfects stamen filaments of rice at the booting stage and transforms kernels into smut balls by utilizing rice nutrients (Tang et al, 2013; Zhang et al, 2014).usually infects spikelets by colonizing the filaments of rice florets, preventing pollen from maturing and then hijacking rice nutrients (Fan et al, 2015, 2020; Yong et al, 2016; Qiu et al, 2019). Moreover, RFS produces ustiloxins and ustilaginoidins that inhibit cell division and represent a threat to human and animal health (Koiso et al, 1994; Li et al, 1995; Meng et al, 2015).

Owing to the genome sequencing and establishing of gene deletion methods in, a number of genes important for pathogenicity inhave been studied (Lü et al, 2016; Zheng et al, 2016; Liang et al, 2018). For example, the effectors SCRE1 and SCRE2 (Fang et al, 2019; Zhang et al, 2020) protein kinases UvPmk1 and UvCDC2 (Tang et al, 2020), transcription factors UvCom1, UvHOX2 and UvPRO1 (Lü et al, 2016; Yu et al, 2019; Chen et al, 2020), apoptotic regulator UvBI-1 (Xie et al, 2019), adenylate cyclase UvAc1 (Guo et al, 2019), phosphodiesterase UvPdeH (Guo et al, 2019), low-affinity iron transport protein Uvt3277 (Zheng et al, 2017), MAP kinase (MAPK) cascade component UvHOG1 (Zheng et al, 2016), and SUN family protein UvSUN2 (Yu M N et al, 2015) were characterized in recent years.

Suppressor of MEK (SMEK) is considered to be a regulatory subunit of protein phosphatase 4 (Yoon et al, 2010). SMEK usually functions as a global regulator by interacting with various intracellular proteins (Kim et al, 2015). In human cells, this regulator protein contributes to the regulation of cell proliferation, cell differentiation, cell cycle, hepatic gluconeogenesis, apoptosis and microtubule organization(Mendoza et al, 2007; Byun et al, 2012; Lyu et al, 2013; Kim et al, 2015, 2017). In invertebrates and protozoa, SMEK was reported to be a regulator of cell development, chemotaxis and longevity (Wolff et al, 2006; Mendoza et al, 2007). In yeast, the ortholog of SMEK1 known as Psy2 interacts with protein phosphatase4-homologous PPH3, and participates in the regulation of cell cycle, DNA repair, drug sensitivity and glucose transport (Gingras et al, 2005; Ma et al, 2014; Omidi et al, 2014; Hustedt et al, 2015).

In the present study, we identified a gene encoding suppressor of MEK null (), the homolog of SMEK1 in mammals and Psy2 in yeast, which was disrupted inmutant A-204. Deletion ofcaused the loss of pathogenicity, abnormal conidiation and disordered conidial germination. The mRNA ofwasspecifically higher during infection, but lower during conidiation. Generally, all evidences indicated that UvSMEK1 was a key regulatorof pathogenicity, conidiation and conidial germination.

RESULTS

Characterization of genes disrupted in mutant A-204

In the preliminary study, we identified a T-DNA insertional mutant A-204 of, which was failed to form smut balls on the inoculated spikelets (Fig. 1-A). To identify the mutated gene in A-204, we performed Southern blotting to determine the copy number of T-DNA inserted into the genome of A-204, and a 1.4-kb hygromycin-resistant cassette was employed as a probe in Southern blotting. The result showed that only one copy of T-DNA was detected in mutant A-204 (Fig. 1-B).

Fig. 1. Identification of mutated gene inT-DNA insertion mutant A-204.

A, Rice false smuts on rice spikelets inoculated with wild-type strain 70-22 and T-DNA insertion mutant A-204.

B, Detection of copy number of T-DNA inserted in the genome of A-204 by Southern blotting.

C, Detection ofexpression via reversed-transcription PCR, andwas employed as a reference gene.

D, Inserted site of T-DNA in the coding region ofinmutant A-204.

E, Conserved SMK-1 domain in UvSMEK1.

F,Phylogenetic analysis of SMEK-1 homologs to UvSMEK-1 in fungi.

The T-DNA flanking regions were amplified by thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). The results showed that T-DNA was inserted into the 5′-coding region of suppressor of MEK1 ortholog (designated as) in the mutant A-204 (Fig. 1-C and -D). Because the 5′-terminal ofcoding region in the reference sequence (GenBank accession number: KDB15555.1) is not complete, we amplified the 5′-terminal ofusing a5′-RACE system, and the sequence ofwas deposited (GenBank accession number: MT884407). We found the amino acid sequence of UvSMEK1 is conserved among filamentous fungi, such as,,,and,and budding yeast. UvSMEK1 is most homologous to cereal pathogenic fungus(Fig. 1-E and -F).

Generation of UvSMEK1 deletion mutants and their complementation

To generatedeletion mutants, the-mediated transformation (ATMT) method was used to transfer the gene replacement cassette into thewild type strain P-1. Two hundred and forty transformants resistant to hygromycin B were picked from the selective medium after culturing at 28oC in darkness for about 7–10 d. Seven of these transformants were confirmed asdeletion mutants (Fig. 2). Among them, the putativedeletion mutants,andwere further confirmed by RT-PCR and DNA sequencing (Fig. S1).

To generatecomplement mutants, the cassettecontaining completegene was transformed intodeletion mutant. Fifty- seven transformants were screened on the 2% TB3 medium with geneticin 418, and transformants,andwere selected and confirmed with PCR, RT-PCR and DNA sequencing (Fig. 2-B, -C and Fig. S1).

UvSMEK1 was essential for pathogenicity and regulates conidiation and conidia germination

deletion mutants,and, and wild type strain P-1 were then used to inoculate rice spikelets at the booting stage. The RFS disease incidence was detected 30 d after inoculation. None of spikelets was infected bydeletion mutants (Fig. 3-A). In contrast, the number of smut balls on spikelets inoculated with the wild type strain P-1 was 13.7 ± 2.7 (Fig. 3-A and Table 1). The complement mutants partially recovered their pathogenicity, with the numbersof smut balls on spikelets inoculated with,andbeing 3.7 ± 2.2, 3.2 ± 1.2 and 4.1 ± 1.9, respectively (Fig. 3-B and Table 1). The expression levels ofat the stages of mycelia growth, conidiation on YT medium, conidiation in YT broth (that also used for aritificial inoculaiton on rice panicle), and 12 h post inoculation (hpi) to 7 d post inoculation (dpi) were determined by qPCR. The results showed that the expression levels ofduring conidiation on YT medium and vegetative growth were not significantly different with those in YT broth; however, the expression levels ofwere significantly higher at 3–7 dpi (Fig. 3-C). This meant the expression ofwas induced by the infection of rice florets. All these findings showed that UvSMEK1 was a critical regulatory element for pathogenicity.

Fig. 2. Deletion ofgene in.

A, Illustration of targeted deletion ofby-mediated transformation (ATMT) and homologous replacement. P11 to P18 represent the primers. UF, Upstream flank of;HYG, Hygromycin resistant gene; DF, Downstream flank of.

B, Illustration of complementation cassette ofgene by ATMT transformation.

C, Reversed-transcription PCR analysis ofdeletion mutants andcomplement mutants. The wild type strain P-1 and transformant Trans-9 with ectopically inserted UF-HYG+-RF cassette were included as controls. M, Marker; a, P-1; b, Trans-9; c,; d,; e,; f,; g,; h,.

Fig. 3. Characterization ofdeletion and complement mutants in.

A, Rice false smut balls on rice spikelets inoculated with wild type strain P-1,deletion mutantand complement mutant.

B, Colonies of P-1,andon potato sucrose agar at 28 oC after 12 d.

C, Expression pattern ofwas determined by quantitative PCR. House-keeping genewas employed as a reference gene. Data are Mean ± SD (= 3). **, Significant difference at the 0.01 level (-test).M, Mycelia; CG, Conidial germination; CS(YTM), Conidial sporulation on yeast extract tryptone (YT) media; CS(YTB), Conidial sporulation in YT broth; hpi, Hours post inoculation; dpi, Days post inoculation.

The percentage of germinated conidia of wild type strain P-1 anddeletion mutants on YT medium was similar (Table 1). However, the conidial size ofdeletion mutants was abnormally larger than that of wild type strain P-1 before germ tube emergence, and also the germ tube ofdeletion mutants was thicker than that of wild type strain andcomplement mutants (Table S1).

We also tested the capacity of conidiation in YT broth and on YT medium. The concentration of conidia produced bydeletion mutants in YT broth was much higher than that of wild type strain P-1 andcomplement mutants (Fig. 4-A and Table S2). In YT broth, the conidia productions ofdeletion mutants and wild type strain P-1 were similar in the period 2–4 dpi, however, conidia production bydeletion mutants was dramatically increased during 5–7 dpi compared to wild type strain P-1. The results were surprisingly different whendeletion mutants and wild type strain P-1 were tested on YT medium. When the conidia were cultured on YT medium, thedeletion mutants lost the capacity of micro-cycle conidiation and produced fewer conidia in the first 3 d (Table 1 and Fig. 4-C). These results hinted at UvSMEK1 functioning as an essential regulator of pathogenicity, conidial germination and conidiation in.

Table 1. Pathogenicity, conidiation and conidial germination in UvSMEK1 deletion and complement mutants.

Fresh mycelia ofstrain/mutants were cultured on potato sucrose agar (PSA) at 28oC for 12 d.The number of rice false smut balls on the inoculated spikelets.Concentration of conidia in yeast extract tryptone (YT) broth was measured after shaking at 130 r/min, 28oC for 7 d.The conidia were cultured on YT media at 28oC for 2 d before observation.

Data are shown as Mean ± SD (= 4). Different letters mark statistically significant differences using the Fisher’s protected least significant difference test (Uppercase letters for< 0.01 and lowercase letters for< 0.05).

Fig. 4. Conidiation, conidial germination and hyphal branching ofdeletion mutant and complement mutant.

A, Log10concentration of conidia produced in yeast extract tryptone (YT) broth in 7 d.

B, Conidia, germination of conidia and conidiation on YT medium ofdeletion mutantand complement mutantafter 3-day culture.

UvSMEK1 deletion mutants exhibited higher tolerance to oxidative, osmotic and cell wall stresses

When cultured on YT medium amended with 0.4 mol/L NaCl, 0.07% H2O2and 0.03% sodium dodecyl sulfate (SDS), the colony diameter of thedeletion mutants was significantly larger than that of the wild type strain P-1 andcomplementary mutants. Moreover, the aerial mycelia ofdeletion mutants that grew on 0.03% SDS were thicker than that of the wild type strain P-1 andcomplement mutants. However, when cultured on YT amended with 100 mg/L Congo red, the colony diameter ofwas similar to that of wild type strain P-1 (Fig. 5 and Table 2). These findings showed that thedeletion mutants were less sensitive to oxidative, osmotic and cell wall stresses than the wild type strain P-1, suggesting UvSMEK1 is also involved in responses to oxidative, osmotic and cell wall stresses.

Fig. 5. Growth ofdeletion and complement mutants in presence of different biotic stresses.

Wild type strain P-1,deletion mutantandcomplement mutantwere cultured on plain yeast extract tryptone (YT) medium or amended with 0.4 mol/L NaCl, 0.07% H2O2, 0.03% sodium dodecyl sulfate (SDS) and 100 mg/L Congo red at 28oC for 15 d.

DISCUSSION

The ATMT method was used to generate T-DNA insertion mutants. To date, UvSUN2, Uvt3277 and UvPRO1 have been disrupted using insertional mutation (Yu M N et al, 2015; Lü et al, 2016; Zheng et al, 2017). Recently, the CRISPR-Cas9 system was used to significantly increase the efficiency of gene replacement by homologous recombination in(Liang et al, 2018; Xu et al, 2019). Considering some genes were still hard to be deleted by CRISPR- Cas9 (personal communication), the ATMT method is still an alternative useful tool for gene deletion and complementation.

Table 2. Responses of mycelial growth of UvSMEK1 deletion and complement mutants to abiotic stress. mm

Fresh mycelia ofstrain/mutants were cultured on media at 28oC for 15 d.

YT, Yeast extract tryptone; SDS, Sodium dodecyl sulfate.

Data are shown as Mean ± SD (= 4). Different letters mark statistically significant differences using the Fisher’s protected least significant difference test (Uppercase letters for< 0.01 and lowercase letters for< 0.05).

Although a few of the genes contributing to pathogenicity have been characterized in rice false smut fungus, regulatory mechanism of pathogenicity needs to be elucidated further. In this study, we characterized the function of UvSMEK1 in. This protein harbors a SMK-1 superfamily domain at the N-terminus. UvSMEK1 is conserved among filamentous fungi and budding yeastHowever, the regulatory function of SMEK1 and its orthologs has not been reported in plant pathogenic fungi.

Sclerotia and chlamydospores ofcan over- winter in paddy fields and serve as the primary infection sources of rice false smut disease (Yu J J et al, 2015; Fan et al, 2016). Both ascospores and chlamydospores germinate to produce huge numbers of secondary conidia, which are considered to be inocula of this disease. Many lines of evidence showed thatmay grow epiphytically on rice leaves and other weed plants, and infect rice spikelets by hyphae or producing conidia at the rice booting stage. In most of the previous studies, the reduced virulence in severalmutants also came with the defects in hyphal growth and conidia production (Xie et al, 2019; Yu et al, 2019; Tang et al, 2020). In the present study, thedeletion mutants exhibited similar hyphal growth but higher production of conidia in YT broth compared to the wild type strain, however, these mutants completely lost their pathogenicity. This suggested that the mechanism of UvSMEK1 regulating pathogenicity inhad a different basis.

In slime mold, SMEK is essential for cell polarity and chemotaxis (Mendoza et al, 2005). The invasive hyphae ofextend along the cell gaps in the floral filaments without penetration, but the infection byblocks the development of ovary and hijacks the rice nutrient supply to the host cell walls (Takano et al, 2006; Tang et al, 2013; Fan et al, 2015). Cell polarity and chemotaxis seem to be very important for the invasion of rice florets by. In filamentous fungi, the switch between isotropic expansion and polar growth is very important for conidial germination and hyphal growth (Knechtle et al, 2003; Guest et al, 2004). The conidia ofdeletion mutants seemed to stay longer in the isotropic expansion before switching to polar growth compared to wild type strain. This finding suggested that UvSMEK1 is a key regulator of switchingbetween isotropic expansion and polar growth. In addition,deletion mutants showed higher tolerance to oxidative, osmotic and cell wall stresses compared with the wild type strain. These clues hint at a defect in polar growth regulation and chemotaxis potentially causing the loss of pathogenicity in.

The abnormal conidiation indeletion mutants suggestedregulates conidiation in. Interestingly,deletion mutants exhibited an increase capacity of conidiation in YT broth, but a reduced capacity of conidiation on YT medium. This phenomenon provided a clue that the regulatory mechanism ofconidiation in liquid vs. solid media is different to some extent, and UvSMEK1 may be at the intersection between two kinds of regulatory networks. Therefore, UvSMEK1 is a critical regulatory element for early-stage rice floret infection and conidiation, making UvSMEK1 a potentialmolecular target for blocking the infection by.

In mammalian cells, suppressor of MEK (SMEK1) is a core regulatory subunit of protein phosphatase 4 (PP4) complex (Yoon et al, 2010). In budding fungus, Psy2 (the orthologue of SMEK1) interacts with protein phosphatase 2A-like protein Pph3 in order to function in regulation (Gingras et al, 2005; Sun et al, 2011). However, little is known about its function in filamentous plant pathogenic fungi. In,protein phosphatase 2A (PP2A) and PP2A-like proteins UvPP2A, UvSIT4 and UvPPG1 were found (data not shown), but the homolog of Pph3 was not detected. Similar situation was reported in another cereal pathogenic fungus(Liu et al, 2018). The mechanism by which UvSMEK1 modulates infection byand other plant pathogenic fungi warrants further studies.

METHODS

Strains, rice variety, plasmids and nucleotide manipulation

A virulent wild typestrain P-1 was used as the starting strain. To test the virulence ofstrains and mutants, a susceptible rice variety Liangyoupeijiu was used in the inoculation experiments. The plasmid pCAMBIA1300 was used for the gene deletion in,and pCAM-NEO constructed in the preliminary study was employed for gene complementation (Table S3).strain AGL-1 was used in the-mediated transformation (ATMT). Southern blotting and TAIL-PCR were performed as described previously (Yu M N et al, 2015).

Phenotypic analysis of U. virens strains and mutants

Thestrain P-1 was routinely cultured on a PSA at 28 oC for 10–15 d (Zheng et al, 2017). The transformants of P-1 were cultured on 2% TB3 (3 g yeast extract, 3 g casamino acids and 2% sucrose) amended with 100 mg/L hygromycin and/or 600 mg/L geneticin 418 (Yu et al, 2015). To determine the pathogenicity ofstrains and mutants, 15 panicles were inoculated by each strain, and the number of false smut balls was counted at 30 d after inoculation. The mixture of conidia and hyphae for inoculation was prepared as described in the previous study (Zheng et al, 2017). We used YT medium and broth to test mycelial growth rate and conidiation capacity of, respectively (Zheng et al, 2016). To test sensitivity of strains to abiotic stress, YT media was amended with 0.05% H2O2(oxidative stress), 0.4 mol/L NaCl (osmotic stress), 0.03% SDS (cell wall stress) and 100 mg/L Congo red (cell wall stress), respectively. The cultures were incubated at 28 oC for 15 d in darkness, and then the colony diameter was measured, and the morphology of the colonies was characterized. The conidiation capacity of the strains was determined using YT broth as described previously (Yu et al, 2019). Four duplicates were performed for each treatment.

Generation of UvSMEK1 deletion mutants

We constructed a gene replacement cassette [upstream flank (UF)-hygromycin resistant gene (HYG)-downstream flank (DF)] ofusing the double-jointed PCR method (Tuorto et al, 2015). This cassette was subcloned into the klenow fragment ofX I-I digested pCAMBIA1300 using a ClonExpress Ultra One Step Cloning Kit (Vazyme, China)to generate gene deletion vector pD-UvSMEK (Tables S3 and S4). The binary vector pD-UvSMEK andstrain AGL-1 were employed in the ATMT transformation ofstrain P-1. The transformants were selected on 2% TB3 amended with 100 mg/L hygromycin (Yu M N et al, 2015). Thedeletion mutants were screened out by detection ofopen reading fragment region and further confirmed by DNA sequencing.

Generation of UvSMEK1 complement mutants

We amplified the complete cassette ofcontaining its promoter, coding region and terminator from genome DNA of wild type strain P-1, and inserted thecassette into the klenow fragment ofR I-I digested pCAM-NEO to generate gene complement vector pC-UvSMEK using a ClonExpress Ultra One Step Cloning Kit (Vazyme, China) (Table S3). The binary vector pC-UvSMEK andstrain AGL-1 were employed in the ATMT transformation ofmutant. The transformants were selected on 2% TB3 amended with 600 mg/L geneticin 418. Thedeletion mutants were screened out by detection ofopen reading fragment region and further confirmed by DNA sequencing.

RT-PCR and qPCR assays

Vegetative mycelia were collected from 2-day-old cultures on YT medium. To stimulate conidiation in, mycelia were cultured in YT broth by shaking (28 oC, 150 r/min) for 3 d. To collect mycelia during infection, the mixture of fresh mycelia and conidia were inoculated into the panicles at the booting stage. The inoculated spikelets were collected at 12 h, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d and 7 d after inoculation. A PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Japan) and SYBRR Premix ExII (Takara, Japan) used to synthesize cDNA and quantitative RT-PCR. Because we prepared the mixture of conidia and mycelia (artificial inocula) fromsamples during conidiation in YT broth, the relative expression level ofat different periods was calculated by the 2-ΔΔCtmethod compared to that of samples during conidiation in YT broth. The(NCBI accession number: KDB17573.1) gene was employed as the reference. Three biological replicates were performed to calculate the mean and the standard deviation. The data obtained from this quantitative PCR assay were subjected to atest wherevalues below 0.05 were considered to be significant. To detect the expression ofin, reverse- transcription PCR was also performed (Table S4).

ACKNOWLEDGEMENTS

This study was supported by the National Key Research and Development Project in China (Grant No. 2016YFD200805), and National Natural Science Foundation of China (Grant Nos. 31301624 and 31571961).

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science; http://www.ricescience.org.

Fig. S1. Detection ofexpression via reversed- transcription PCR.

Table S1. Size and length-width ratio ofdeletion/ complementmutants in yeast extract tryptone broth.

Table S2. Concentration of conidia produced bydeletion/complementmutants in yeast extract tryptone broth.

Table S3. Strains and vectors used in this study.

Table S4. Primers used in this study.

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14 September 2020;

12 January 2021

Liu Yongfeng (liuyf@jaas.ac.cn)

Copyright ? 2021, China National Rice Research Institute. Hosting by Elsevier B V

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Peer review under responsibility of China National Rice Research Institute

http://dx.doi.org/10.1016/j.rsci.2021.07.006

(Managing Editor: Wang Caihong)