bZIP Transcription Factor UvATF21 Mediates Vegetative Growth, Conidiation, Stress Tolerance and Is Required for Full Virulence of Rice False Smut Fungus Ustilaginoidea virens

Liu Yueran, Qu Jinsong, Wang Yufu, Yin Weixiao, Luo Chaoxi

Research Paper

bZIP Transcription Factor UvATF21 Mediates Vegetative Growth, Conidiation, Stress Tolerance and Is Required for Full Virulence of Rice False Smut Fungus

Liu Yueran#, Qu Jinsong#, Wang Yufu, Yin Weixiao, Luo Chaoxi

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bZIP proteins are widely distributed in eukaryotic organisms and regulate a diverse range of physiological processes. Several bZIP proteins have previously been identified in. However, the biological roles of these bZIP proteins in this pathogen are still unknown. Here, one of these bZIP protein coding genes,, was functionally characterized. Targeted deletion ofresulted in reduced conidiation and pathogenicity despite of the increased vegetative growth. The deletion mutants also significantly decreased the sensitivity to osmotic and oxidative stresses. Interestingly, deletion ofexhibited different performances to cell wall integrity stress. These results indicated thatplayed crucial roles in vegetative growth, conidiation, stress response, and full virulence in..

; bZIP protein; conidiation; stress response; pathogenicity

Transcription factors (TFs) recognize specific DNA sequences to regulate the expression of genes involved in the cellular and developmental process (Warren, 2002; Lambert et al, 2018; Gundu et al, 2020). As one of the most widely distributed and conserved TF proteins in eukaryotes, the bZIP (basic leucine zipper) proteins contain the basic and leucine-zipper region which mediates sequence-specific DNA binding and is required for dimerization (Hurst, 1995; Fassler et al, 2002; Dr?ge-Laser et al, 2018; Lorenzo, 2019).

In filamentous fungi or oomycetes, the bZIP proteins are involved in many critical processes, including development (Kong et al, 2015), amino acid biosynthesis (Tang et al, 2015), unfolded protein response (Tang et al, 2015), nutrient utilization (Amich et al, 2013), and various stress responses (Tian et al, 2011). The activating transcription factor (ATF) family representing a large group of bZIP proteins has been studied in fungi (Leiter et al, 2021). The CPTF1 from(Nathues et al, 2004), MoAtf1 from(Guo et al, 2010), and FgATF1from(Jiang et al, 2015) play essential roles in oxidative stress response and virulence. ATF-2 is phosphorylated in mammalian cells by mitogen-activated protein kinases (MAPK) stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK), and p38 (Gupta et al, 1995), indicating ATF proteins are downstream of MAPK pathway. Atfl in yeast is targeted by MAPK Styl (Wilkinson et al, 1996). ATF-1 acts as the downstream of MAPK and regulates the expressions ofandgenes involved in conidiation and response to fludioxonil in(Yamashita et al, 2008). In, AtfA physically interacts with the MAPK SakA to regulate oxidative stress responses, development and spore functions (Lara-Rojas et al, 2011). In addition to oxidative stress response, ATF1 is also involved in other stress tolerance. Even though thefromdoes not contribute to the resistance to oxidative stress, deletion ofenhances the sensitivity to sodium dodecyl sulphate, amphotericin B and tert-butyl hydroperoxide (Nimmanee et al, 2014). In, the VdAtf1 negatively regulates nitric oxide tolerance and plays an essential role in the formation of penetration peg by regulatingexpression (Tang et al, 2020a, b). Additionally, CsAtf1 is involved in fludioxonil sensitivity and virulence of(Song et al, 2022).

Fig. 1. Phylogenetic analysis of UvATF21 and sequence alignments of bZIP_ATF2 domain.

A, Phylogenetic analysis of UvATF21. Phylogenetic tree was constructed using MEGA7. GenBank accession numbers are indicated in brackets.

B, Alignment of bZIP_ATF2 domain sequences from different genera.

The sequences were downloaded from the NCBI website (https://www.ncbi.nlm.nih.gov/).

Rice false smut caused by the fungusis one of the most destructive rice diseases in rice-growing regions worldwide. The typical symptom ofis the generation of false smut balls that vary from yellowish-orange to greenish-black in rice panicles. A number of TFs inhave been studied in recent years, such as UvHOX2 (Yu et al, 2019), UvCom1 (Chen et al, 2020), UvCGBP1 (Chen et al, 2021), UvZnFTF1 (Song et al, 2021) and UvMsn2 (Xu et al, 2021). Treatment with NaCl enhances the nucleus localization of UvMsn2, and deletion ofreduces growth, conidiation and pathogenicity in the rice false sweat fungus (Xu et al, 2021). In addition, UvCGBP1 is involved in the development and virulence via regulating the expression ofand, both of which are MAPK-related genes (Chen et al, 2021).

Previously, we have identified bZIP proteins in. However, the functions of these proteins still need to be explored. In this study, we investigated the potential roles of UvATF21(KDB18880), one of the bZIPs in. The phylogeny and transcriptional pattern ofwere also analyzed. Furthermore, we characterized the function ofand found thatwas a negative regulator for vegetative growth but was required for the asexual development, oxidative stress response, and pathogenicity in.

Fig. 2. Expression level ofat different stages.

Expression ofwas quantified using qRT-PCR, with the synthesis of cDNA from each sample, including mycelium, 1, 3, 5, 7, 9, 11 and 13 d post inoculation (dpi). Relative transcript level at the mycelium stage was regarded as the reference and normalized to 1. Data are Mean ± SD (= 3). Different lowercase letters above the bars represent statistical significance at the 0.05 level.

RESULTS

Phylogenetic analysis of UvATF21

Previously, we have identified several bZIP proteins from(Yin et al, 2017), whose functions still remain to be investigated. One of these proteins, KDB18880, was selected for further research. This protein was predicted to contain bZIP_ATF2 domain and identified as an ortholog of ATF21 proteins from other genera (Fig. 1-A). Therefore, it was named UvATF21. The ATF proteins from different fungi were selected for phylogenetic analysis and were divided into two groups (Fig. 1-A). Several proteins, including UvATF21, were clustered into the first group, indicating that the orthologs of UvATF21 existed in a few genera. The proteins in the second group had both bZIP_ATF1 and bZIP_ATF2 domains. Multiple sequence alignment of UvATF21 and its homologs showed that the bZIP_ATF2 domain was well conserved (Fig. 1-B).

Transcriptional pattern analysis of UvATF21

To gain insights into the possible function ofin, its expression levels at different stages were analyzed using qRT- PCR during the mycelia and various post-inoculation stages. The results showed thatexhibited the highest expression level at 5 d post inoculation (dpi) (Fig. 2).

UvATF21 negatively regulates vegetative growth

To investigate the potential roles of, the gene deletion mutants were generated using a combination of homologous recombination and CRISPR-Cas9 systems. The deletion mutants were verified using different primer pairs (Fig. S1), and one of them was selected to obtain complemented strain (Fig. S2). The expression levels ofin wild type (WT, JS60-2), knock-out mutants (?and ?), and complemented strain (?) were detected by semi-quantitative RT-PCR (Fig. S3).

Fig. 3.negatively regulates vegetative growth.

A, Colony morphology of strains for 14 d after culturing on PSA, PDA and YTD media.

B, Colony diameter of each strain in A was measured.

C, Dry weights of strains measured at 7 d after culturing in PSB medium.

Data in B and C are Mean ± SD (= 3). The asterisks represent statistical significance (< 0.05).

JS60-2, Wild type strain of; ?and, Knock-out mutants of; ?, Complemented strain of.PSA, 200 g potato, 20 g sucrose and 15 g agar; PSB, 200 g potato and 20 g sucrose; PDA, 200 g potato, 20 g glucose and 15 g agar; YTD, 1 g yeast powder, 1 g peptone, 10 g glucose and 15 g agar.

WT, knock-out mutants and complemented strain were incubated on PSA (200 g potato, 20 g sucrose, and 15 g agar), YTD (1 g yeast powder, 1 g peptone, 10 g glucose, and 15 g agar) and PDA (200 g potato, 20 g glucose, and 15 g agar) media, to investigate whetherwas involved in mycelial growth. Compared with WT and complemented strain, the knock-out mutants showed a higher growth rate on PSB (200 g potato and 20 g sucrose) and PDA media, but no difference was observed on YTD media (Fig. 3-A and -B). In addition, the dry weight of mycelium cultured on PSB media was measured, and consistent results were observed, which showed that the mutants were increased in dry weight in compared with the WT and complemented strain (Fig. 3-C). These results indicated thatnegatively regulates vegetative growth.

UvATF21 regulates conidiation but not conidial germination

The conidia play an indispensable role ininfection. To investigate whetheris involved in conidiation, the strains ofwere cultured on PSB for 7 d, and the number of conidia was measured. The morphology and germination rate of the conidia were also observed, but there was no discernible difference (Figs. 4-A and S4). The number of conidia in mutants was significantly decreased compared with WT and complemented strain (Fig. 4-B). These results implied thatregulates the conidiation, but not conidial morphology and germination.

UvATF21 is involved in stress tolerance

To investigate whether the loss ofleads to growth defects under different stress conditions, the growth of mutants in the presence of various stresses were observed. The mutants showed lower inhibition rates on the PSA media containing NaCl and sorbitol compared with WT and complemented strain (Fig. 5), indicating thatnegatively regulates the osmotic stress response of. The mycelial growth was also measured on media containing sodium dodecyl sulfate (SDS), calcofluor white (CFW), and Congo red (CR) to examine the cell wall integrity. The inhibition rates of mutants were significantly lower than those of WT and complemented strain under SDS and CR conditions. Nonetheless, the mutants were more sensitive to CFW (Fig. 5). These results indicated thatplays a crucial role in cell wall integrity. Moreover, the strains were inoculated on PSA containing H2O2to investigate whetheris involved in the oxidative stress response. The mutants were less sensitive to H2O2than WT and complemented strain (Fig. 5). This result indicated thatis a negative regulator of the oxidative stress response in. Taken together, these results suggested thatis involved in stress tolerance.

Fig. 4.contributes to conidiation.

A, Morphology of conidia produced by the strainsof.

B, Statistical analysis of the conidiation.

The strains were cultured in PSB (200 g potato and 20 g sucrose) media for 7 d. Data are Mean ± SD (= 3). The asterisks represent statistical significance (< 0.05).

JS60-2, Wild type strain of; ?and, Knock-out mutants of; ?, Complemented strain of.

UvATF21 is required for full virulence

To explore whethercontributes to pathogenicity, the conidial and hyphal suspensions of the WT, mutants and complemented strain were infiltrated into rice panicles, and the false smut balls were measured at 21 dpi. The numbers of false smut balls on rice plants inoculated with the mutants were remarkably decreased in contrast to WT and complemented strain (Fig. 6), indicating thatlikely contributes to the full virulence of.

DISCUSSION

Reactive oxygen species (ROS) play a central role in plant signaling and development processes (Mhamdi and van Breusegem, 2018; Waszczak et al, 2018). In plant-pathogen interactions, plants fight off invading pathogens with a transient burst of ROS (Qi et al, 2017). Thus, tolerance to oxidative stress is essential for the successful infection of pathogens. It was previously revealed that many ATF proteins are involved in coping with ROS stress. In,contributes to the full virulence by mediating oxidative stress responses (Guo et al, 2010). Theinis required for oxidative stress tolerance (Sakamoto et al, 2008). Themutant ofwas sensitive to some stresses but not hydrogen peroxide (Nimmanee et al, 2014). Althoughdoes not function in response to oxidative stress, its deletion strains are more resistant to nitrosative stress (Fang et al, 2017; Tang et al, 2020a).has a limited impact on stress tolerance (Temme et al, 2012). In this study, deletion ofled to augmented tolerance to oxidative and other stresses except CFW, to which it was insensitive. These results suggested thatplays crucial roles in stress response. Although the conidiation was compromised, there was no significant difference of germination rate on WA (water agar) or PSA. The reason of the decreased virulence is probably that the conidia generated bymutants are less tolerant to various environmental stresses when infecting rice.

Fig. 5.is involved in stress tolerance.

A, Colony morphology ofcultured on the media of PSA (200 g potatoes, 20 g sucrose and 15 g agar) containing 0.5 mol/L NaCl, 0.9 mol/L sorbitol, 0.04% sodium dodecyl sulfate (SDS), 200 μg/mL Congo red (CR), 120 μg/mL calcofluor white (CFW) and 0.03% H2O2for 14 d.

B, Mycelial growth inhibition rates were calculated. Data are Mean ± SD (= 3). The asterisksrepresent statistical significance (< 0.05).

JS60-2, Wild type strain of; ?and, Knock-out mutants of; ?, Complemented strain of.

Fig. 6.contributes to pathogenicity.

A, Rice panicles were inoculated, and the infected panicles were examined and photographed at 21 d post inoculation (dpi).

B, The number of false smut balls per rice panicle after 21 dpi. Data are Mean ± SD (= 12). The asterisks represent statistical significance (< 0.05).

JS60-2, Wild type strain of; ?and, Knock-out mutants of; ?, Complemented strain of.

The bZIP proteins regulate the development of filamentous fungi or oomycetes, thereby mediating vegetative growth. The mutants ofinexhibit reduced vegetative growth and different colony morphologies (Shin et al, 2020). In, themutants exhibit defects in aerial hyphal growth, yeast-to-hypha transition, and conidia (Song et al, 2018). Knock-out ofled to increased vegetative growth, indicating thatplays a negative role in vegetative growth.

The ATF/CREB family transcriptional factor Atf1 is regulated by stress-activated MAPKin yeast (Shiozaki and Russell, 1996;Wilkinson et al, 1996). The bindingsite is defined as TGACGTCA (Hai, 2006). AtfA interacts with MAPK SakA to regulate stress response, development, and spore functions of(Lara-Rojas et al, 2011). In addition,contributes to nitric oxide resistance and pathogenicity via regulating the expression of, the promoter of which contains a conserved ACGTA(A/T)TG motif (Tang et al, 2020a). Therefore, for further research on UvATF21, one important thing is to identify the interacted proteins in search of the signaling pathway in the upstream. It will also be interesting to identify genes that are directly regulated by UvATF21.

In conclusion, we identified that one of the bZIP proteins, UvATF21, is involved in the development and pathogenicity in. Deletion ofincreases growth but decreases conidiation and pathogenicity.is required for some stress tolerance, including H2O2, indicating thatplays potential roles in these processes. Our results give insights into the virulence mechanism ofand provide reference for related studies in other fungal systems.

METHODS

Strain and culture medium

Wild-type strain JS60-2 ofwas cultured at 27oC with 160 r/min. PSB, PSA, PDA and YTD were used to measure phenotype.

UvATF21 protein sequence and phylogenetic analysis

gene and the other orthologous sequences were downloaded from NCBI website (https://www.ncbi.nlm.nih.gov/), and the conserved domain database structure was used to predict the protein domain. The MEGA7 adjacency method (neighbor-joining, NJ) was used for phylogenetic tree analysis.

Analysis of UvATF21 gene expression

Rice spikelets were collected at 1, 3, 5, 7, 9, 11 and 13 dpi, and the RNA of these spikelets and mycelium were extracted. The cDNA obtained by reverse transcription was detected for gene expression using the CFX96 real-time PCR system (Bio-RAD Laboratories Inc, CA, USA). Statistical significance analysis was performed by SPSS software.

UvATF21 knock-out transformants and recovery strains obtained

The gene deletion was performed using the method described byLiang et al (2018) and Xie et al (2019). The Crispr- UvATF21F and Crispr-UvATF21R primers were annealed and inserted into pCas9:tRp-gRNA vector. The double-joint PCR amplified the homologous recombinant fragments. The knock-out efficiency will be increased when additional CRISPR/Cas9 vector was added. Through polyethylene glycol mediated protoplast transformation, the CRISPR/Cas9 vector plasmid and the upstream and downstream flanking sequence ofwere simultaneously introduced into the protoplasts of WT strains for gene knock-out (Zheng et al, 2016). The fragment was amplified using UvATF21-CF and UvATF21-CR primers and inserted into a complemented vector pKNRG823. The complemented vector was transformed into knock-out strains by protoplast transformation. The knock-out and complemented transformants were screened on PSA medium containing 100 μg/mL hygromycin or 200 μg/mL geneticin. Semi-quantitative PCR and qRT-PCR were performed for further verification. Primers used in this study were listed in Table S1.

Determination of colony morphology, growth rate and dry weight

The plugs of fungus were cultured on PSA, PDA and YTD plates, respectively. The colony morphology was observed, and the colony size was measured by the cross-crossing method after 14 d. The mycelia were collected after 7 d cultured in 50 mL PSB and dried at 50oC for 3 d to measure the dry weight. Each strain had three replicates and at least three independent replicates in this study.

Determination of conidia production, conidia morphology and germination rate

After 7 d cultured in 50 mL PSB, the conidial suspension was collected by filtering through two layers of gauze, and the contributions were counted by blood counting plate. The conidium suspension was centrifuged at 7 000 r/min for 5 min, the supernatant was discarded, and the precipitate was suspended using sterile water with the final concentration of 1′106conidia/mL. The 100 μL conidia suspension was spread on PSA or WA (15 g agar) plates and cultured in the dark at 27 oC. Germination rates were measured after 12 h and 24 h, respectively. Each strain had three replicates and at least three independent repetitions.

Determination of sensitivity to different stresses

The plugs of fungus were cultured on PDA containing 0.5 mol/LNaCl, 0.9 mol/L sorbitol, 0.04% SDS, 200 μg/mL CR, 120 μg/mL CFW and 0.03% H2O2, respectively. After 14 d, the colony size was measured. The inhibition rates were calculated as follows: Inhibition rate = (Average of strain colony diameters on PSA – Average of strain colony diameters on PSA with a chemical added) / Average of strain colony diameters on PSA × 100%, and it should minus the plug diameter when the colony diameter was measured.

Determination of virulence

The strains were cultured on 50 mL PSB medium for 7 d. The conidial concentration of each strain was adjusted to 2 × 106conidia/mL. The medium containing mycelia and conidia was broken with a high-speed blender, and 2 mL mixture was injected into a single rice panicle at the booting stage and cultured in a greenhouse at 27oC with a relative humidity of 90%–100%. Then, the rice was placed in the greenhouse at 27 oC with 80% relative humidity, and the number of false smut balls was counted at 21 dpi. Each strain inoculated at most minuscule 12 spikes, and at least three independent replicates were performed.

Data statistics and analysis

Data statistics and mapping were completed by Microsoft Office 2019. The standard deviation between repetitions was calculated as the value of the error line on the bar chart, and one-way analysis of variance was performed by the SPSS software.

ACKNOWLEDGEMENTS

This study was supported by the National Natural Science Foundation of China (Grant No. 31701736), Key Research and Development Program of Hubei Province, China (Grant No. 2021BBA236), and National Key Research and Development Program, China (Grant No. 2016YFD0300700).

SUPPLEMENTAL DATA

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

Fig. S1. Target knock-out strategy and transformant verification of.

Fig. S2. Verification ofcomplemented transformants.

Fig. S3. Detection ofexpression in knock-out mutants and complemented strain.

Fig. S4. Germination of conidia of.

Table S1. Primers used in this study.

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