Autophagy-Related Gene PlATG6a Is Involved in Mycelial Growth, Asexual Reproduction and Tolerance to Salt and Oxidative Stresses in Peronophythora litchii

Autophagy is ubiquitously present in eukaryotes. During this process, intracellular proteins and some waste organelles are transported into lysosomes or vacuoles for degradation, which can be reused by the cell to guarantee normal cellular metabolism. However, the function of autophagy-related (ATG) proteins in oomycetes is rarely known. In this study, we identified an autophagy-related gene, PlATG6a, encoding a 514-amino-acid protein in Peronophythora litchii, which is the most destructive pathogen of litchi. The transcriptional level of PlATG6a was relatively higher in mycelium, sporangia, zoospores and cysts. We generated PlATG6a knockout mutants using CRISPR/Cas9 technology. The P. litchii Δplatg6a mutants were significantly impaired in autophagy and vegetative growth. We further found that the Δplatg6a mutants displayed decreased branches of sporangiophore, leading to impaired sporangium production. PlATG6a is also involved in resistance to oxidative and salt stresses, but not in sexual reproduction. The transcription of peroxidase-encoding genes was down-regulated in Δplatg6a mutants, which is likely responsible for hypersensitivity to oxidative stress. Compared with the wild-type strain, the Δplatg6a mutants showed reduced virulence when inoculated on the litchi leaves using mycelia plugs. Overall, these results suggest a critical role for PlATG6a in autophagy, vegetative growth, sporangium production, sporangiophore development, zoospore release, pathogenesis and tolerance to salt and oxidative stresses in P. litchii.


Introduction
Oomycetes are a class of ubiquitous filamentous eukaryotic microorganisms, which are evolutionarily close to photosynthetic algae [1]. Many oomycetes are economically significant pathogens affecting agriculture, forestry and the ecosystem. For example, Phytophthora infestans, P. sojae, P. capsici and Peronophythora litchii severely damage potato, soybean, cucurbits, and litchi, respectively [2,3]. Among them, litchi downy blight caused by P. litchii is the most destructive disease of litchi, leading to 30% to 80% yield losses annually [3]. However, the molecular mechanisms of P. litchii growth, development and pathogenesis are largely unknown [4].
By BLAST searching using PsAtg6a sequence as a bait, we identified an Atg6a ortholog in P. litchii, hereinafter named PlAtg6. We searched the orthologs of PlATG6a in oomycete species, including P. sojae, P. capsici, Pythium ultimum and Hyaloperonospora parasitica. Orthologs of PlATG6a could be found in all these species (Supplementary Table S2), indicating that PlATG6a orthologs are widespread in oomycetes ( Figure 1A).
Transcriptional analysis showed that the PlATG6a transcripts were significantly upregulated in zoospores (ZOs) and cysts (CYs), and down-regulated during infection compared with mycelium (MY) ( Figure 1B). The expression profile of PlATG6a was dramatically different from that of PsATG6a, which was up-regulated during infection. These results suggest that PlATG6a might function in the growth and asexual development stages. In this study, we focused on the function of PlATG6a.  [23] with PlActin gene as the internal control. The MY value was set as "1". Asterisks indicate significant difference compared with MY (** p < 0.01, ttest). These experiments were repeated three times.
Transcriptional analysis showed that the PlATG6a transcripts were significantly upregulated in zoospores (ZOs) and cysts (CYs), and down-regulated during infection compared with mycelium (MY) ( Figure 1B). The expression profile of PlATG6a was dramatically different from that of PsATG6a, which was up-regulated during infection. These results suggest that PlATG6a might function in the growth and asexual development stages. In this study, we focused on the function of PlATG6a. (B) Expression pattern of PlATG6a during the asexual life cycle and infection stages was analyzed by quantitative reverse transcription PCR (qRT-PCR). MY: mycelia; SP: sporangia; ZO: zoospore; CY: cyst; GC: germination of cyst; hpi: hours post inoculation. Relative expression levels were calculated using the 2 −∆∆CT method [23] with PlActin gene as the internal control. The MY value was set as "1". Asterisks indicate significant difference compared with MY (** p < 0.01, t-test). These experiments were repeated three times.

Deletion of PlATG6a Affected Autophagy in P. litchii
To investigate the function of PlATG6a, we generated a deletion mutant of PlATG6a in P. litchii using CRISPR/Cas9 technology. Two single-guide RNAs were designed to disrupt the PlATG6a coding region (Figure 2A). The transformants were screened by G418 resistance and then verified by genomic PCR and sequencing ( Figure 2B,C). Finally, two ∆platg6a mutants (T32, T47) were obtained, and a transformant that failed to delete the PlATG6a gene was selected as the control (CK). We also found that transcription of PlATG6a was undetected in T32 and T47, while it was detectable in the WT (wild-type strain, SHS3) and CK strains ( Figure 2D), confirming successful deletion of this gene in T32 and T47 strains. In P. litchii, we observed more fluorescence in starved mycelia using monodansylcadaverine (MDC) staining ( Figure S1), suggesting that autophagy occurred under starvation. Compared to WT, the ∆platg6a mutants showed a decreased accumulation of autophagosomes, as visualized by MDC staining, in hyphal cells ( Figure 2E). Deletion of PlATG6a also resulted in a significant transcriptional change of more than half the ATG genes ( Figure S2). These results suggest that the autophagy pathway was affected in the ∆platg6a mutants.
in P. litchii using CRISPR/Cas9 technology. Two single-guide RNAs were designed to disrupt the PlATG6a coding region (Figure 2A). The transformants were screened by G418 resistance and then verified by genomic PCR and sequencing ( Figure 2B,C). Finally, two Δplatg6a mutants (T32, T47) were obtained, and a transformant that failed to delete the PlATG6a gene was selected as the control (CK). We also found that transcription of PlATG6a was undetected in T32 and T47, while it was detectable in the WT (wild-type strain, SHS3) and CK strains ( Figure 2D), confirming successful deletion of this gene in T32 and T47 strains. In P. litchii, we observed more fluorescence in starved mycelia using monodansylcadaverine (MDC) staining ( Figure S1), suggesting that autophagy occurred under starvation. Compared to WT, the Δplatg6a mutants showed a decreased accumulation of autophagosomes, as visualized by MDC staining, in hyphal cells ( Figure 2E). Deletion of PlATG6a also resulted in a significant transcriptional change of more than half the ATG genes ( Figure S2). These results suggest that the autophagy pathway was affected in the Δplatg6a mutants.  Table  S1). (D) Transcription of PlATG6a was analyzed by qRT-PCR in wild-type (WT) WT, CK and  Table S1). (D) Transcription of PlATG6a was analyzed by qRT-PCR in wild-type (WT) WT, CK and ∆platg6a mutants (T32 and T47). "***" indicates significant difference compared with WT (p < 0.001, t-test). These experiments were repeated three times. (E) Visualization of autophagosome by MDC staining. The WT and ∆platg6a mutants were incubated in carrot juice agar (CJA) medium for 3 days. After 3 washes and incubation with sterile distilled water for 4 h, hyphae samples were stained with MDC and analyzed by microscopy. Bars = 10 µm. WT: wild-type strain; CK: the transformant failed to acquire PlATG6a mutation (control).

PlATG6a Is Required for Normal Hyphal Growth, Sporangium Production and Zoospore Release
To characterize the function of PlATG6a in P. litchii growth and differentiation, we first inoculated WT, CK and the ∆platg6a mutants on CJA medium and analyzed their colony morphology and hyphal growth at 5 days post inoculation (dpi). The ∆platg6a mutants showed significantly reduced growth rate on the CJA medium compared with that of WT ( Figure 3A,B). This result indicates that PlATG6a is associated with vegetative growth in P. litchii.

PlATG6a Is Required for Normal Hyphal Growth, Sporangium Production Release
To characterize the function of PlATG6a in P. litchii growth and dif first inoculated WT, CK and the Δplatg6a mutants on CJA medium and colony morphology and hyphal growth at 5 days post inoculation (dpi). T tants showed significantly reduced growth rate on the CJA medium com of WT ( Figure 3A,B). This result indicates that PlATG6a is associated growth in P. litchii. In its asexual life cycle, P. litchii produces sporangia, which then rel Sporangia and zoospores play essential roles in both the initial stage of in spread of oomycetes from host to host [4,24]. To investigate the function asexual development, we collected and calculated the number of sporangi mutants, CK and WT grown on CJA medium at 25 °C for 5 days. We fo PlATG6a significantly impaired the production of sporangia (Figure 4 A affect sporangia morphology ( Figure 4C,D). Zoospore release occurred w the sporangia in water, and the rate was measured at 1 and 2 h post incuba showed that Δplatg6a mutants released more zoospores compared with W 1 h after incubation (Figure 4 E,F). Deletion of PlATG6a did not affect c rate (Figure 4 G). These results indicate that PlATG6a affected the sporang and zoospore release. (B) The growth rate was measured and calculated. Bar chart depicts the mean ± SD. Asterisks represent significant difference compared with WT (** p < 0.01, n = 9, t-test).
In its asexual life cycle, P. litchii produces sporangia, which then release zoospores. Sporangia and zoospores play essential roles in both the initial stage of infection and the spread of oomycetes from host to host [4,24]. To investigate the function of PlATG6a in asexual development, we collected and calculated the number of sporangia of the ∆platg6a mutants, CK and WT grown on CJA medium at 25 • C for 5 days. We found that loss of PlATG6a significantly impaired the production of sporangia ( Figure 4A,B), but did not affect sporangia morphology ( Figure 4C,D). Zoospore release occurred when incubating the sporangia in water, and the rate was measured at 1 and 2 h post incubation. Our results showed that ∆platg6a mutants released more zoospores compared with WT, especially at 1 h after incubation ( Figure 4E,F). Deletion of PlATG6a did not affect cyst germination rate ( Figure 4G). These results indicate that PlATG6a affected the sporangium production and zoospore release.

PlATG6a Was Involved in the Morphology of Sporangiophore
To further investigate the mechanism of PlATG6a's involvement in sporangium production, we next assessed the morphological characteristic of the Δplatg6a mutants. We found that each sporangiophore of the Δplatg6a mutants produced fewer branches, as compared with WT and CK strains ( Figure 5). The result suggests that PlATG6a affects sporangium production, likely via contributing to the formation of sporangiophore branches.

PlATG6a Was Involved in the Morphology of Sporangiophore
To further investigate the mechanism of PlATG6a's involvement in sporangium production, we next assessed the morphological characteristic of the ∆platg6a mutants. We found that each sporangiophore of the ∆platg6a mutants produced fewer branches, as compared with WT and CK strains ( Figure 5). The result suggests that PlATG6a affects sporangium production, likely via contributing to the formation of sporangiophore branches.

Virulence of Δplatg6a Mutants
We next tested the virulence of the Δplatg6a mutants, CK and WT using detached litchi leaves. There was no significant difference in virulence between the Δplatg6a mu tants and WT or CK strains when litchi leaves were inoculated with zoospores of these strains ( Figure 6A,B). In fungi, laccases participate in the oxidation of antibiotics such as flavonoids and phytoalexins, and thus contribute to the virulence of pathogens [25]. In oomycetes, laccase activity is also associated with plant infection [26][27][28]. Here we also tested the laccase activity of the Δplatg6a mutants T32 and T47 and found that they showed similar laccase activity as compared with WT and CK ( Figure S3). Therefore, we conclude that loss of PlATG6a did not affect the virulence of P. litchii zoospores, which displayed normal laccase activity.

Virulence of ∆platg6a Mutants
We next tested the virulence of the ∆platg6a mutants, CK and WT using detached litchi leaves. There was no significant difference in virulence between the ∆platg6a mutants and WT or CK strains when litchi leaves were inoculated with zoospores of these strains ( Figure 6A,B). In fungi, laccases participate in the oxidation of antibiotics such as flavonoids and phytoalexins, and thus contribute to the virulence of pathogens [25]. In oomycetes, laccase activity is also associated with plant infection [26][27][28]. Here we also tested the laccase activity of the ∆platg6a mutants T32 and T47 and found that they showed similar laccase activity as compared with WT and CK ( Figure S3). Therefore, we conclude that loss of PlATG6a did not affect the virulence of P. litchii zoospores, which displayed normal laccase activity. However, the Δplatg6a mutants produced fewer sporangia, which are reported to be important for initial infection [24]. Reduced virulence could be expected if we were to inoculate litchi leaves with mycelial plugs, because the Δplatg6a mycelial plugs carried fewer sporangia. Our results showed that the lesion length caused by the Δplatg6a mycelial plugs was reduced, compared with those caused by WT or CK mycelial plugs ( Figure  6 C,D). Therefore, PlATG6a is involved in plant infection, likely via regulating asexual development.

PlATG6a Is Involved in Tolerance to Salt and H2O2 Stress
To investigate whether PlATG6a is related to tolerance to salt stress in P. litchii, the Δplatg6a mutants, WT and CK strains were grown on Plich medium supplemented with 0.05 M NaCl or 0.1 M CaCl2 for 7 days, before measurement of the colony diameter. The growth inhibition rates of the Δplatg6a mutants were significantly (p < 0.01) higher than those of WT or CK strains ( Figure 7A,B), suggesting that PlATG6a may function in tolerance to salt stress. However, the ∆platg6a mutants produced fewer sporangia, which are reported to be important for initial infection [24]. Reduced virulence could be expected if we were to inoculate litchi leaves with mycelial plugs, because the ∆platg6a mycelial plugs carried fewer sporangia. Our results showed that the lesion length caused by the ∆platg6a mycelial plugs was reduced, compared with those caused by WT or CK mycelial plugs ( Figure 6C,D). Therefore, PlATG6a is involved in plant infection, likely via regulating asexual development.

PlATG6a Is Involved in Tolerance to Salt and H 2 O 2 Stress
To investigate whether PlATG6a is related to tolerance to salt stress in P. litchii, the ∆platg6a mutants, WT and CK strains were grown on Plich medium supplemented with 0.05 M NaCl or 0.1 M CaCl 2 for 7 days, before measurement of the colony diameter. The growth inhibition rates of the ∆platg6a mutants were significantly (p < 0.01) higher than those of WT or CK strains ( Figure 7A,B), suggesting that PlATG6a may function in tolerance to salt stress.
In C. neoformans, the atg6∆ mutant was more sensitive to H 2 O 2 treatment [19]. Peroxides are the signature products of the earliest defense responses of plants and play an important role in plant immunity against pathogens [29]. Here, we tested the sensitivity of the ∆platg6a mutants, using the CK and WT strains as the control. The results ( Figure 8A,B) showed that the ∆platg6a mutants were significantly hypersensitive to the oxidative stress caused by H 2 O 2 treatment, as compared to the WT or CK strains. supplement. Images were taken at 7 dpi. (B) Colony diameter was measured at 7 dpi. Growth inhibition rate (%) was calculated. Mean ± SD (n = 9 for each strain). Asterisks denote significant differences vs. WT (* p < 0.05; ** p < 0.01; t-test).
In C. neoformans, the atg6Δ mutant was more sensitive to H2O2 treatment [19]. Peroxides are the signature products of the earliest defense responses of plants and play an important role in plant immunity against pathogens [29]. Here, we tested the sensitivity of the Δplatg6a mutants, using the CK and WT strains as the control. The results ( Figure  8A,B) showed that the Δplatg6a mutants were significantly hypersensitive to the oxidative stress caused by H2O2 treatment, as compared to the WT or CK strains.  supplement. Images were taken at 7 dpi. (B) Colony diameter was measured at 7 dpi. Growth inhibition rate (%) was calculated. Mean ± SD (n = 9 for each strain). Asterisks denote significant differences vs. WT (* p < 0.05; ** p < 0.01; t-test). In C. neoformans, the atg6Δ mutant was more sensitive to H2O2 treatment [19]. Peroxides are the signature products of the earliest defense responses of plants and play an important role in plant immunity against pathogens [29]. Here, we tested the sensitivity of the Δplatg6a mutants, using the CK and WT strains as the control. The results ( Figure  8A,B) showed that the Δplatg6a mutants were significantly hypersensitive to the oxidative stress caused by H2O2 treatment, as compared to the WT or CK strains.  . Data are mean ± SD (n = 9). Asterisks represent significant differences vs. WT (** p < 0.01) based on t-test. We next assessed the expression level of PlATG6a under oxidative stress, at different time points post exposure to H 2 O 2 , following the established method [15,30,31]. The result showed that transcription of PlATG6a was significantly up-regulated at 5 to 55 min after H 2 O 2 treatment ( Figure 8C), implying that this gene plays a role in response to oxidative stress. Furthermore, we examined the expression level of five selected peroxidase-encoding genes [31] in the WT strain and the ∆platg6a mutant. Four out of the five tested genes displayed a higher expression level in the WT strain than that in the mutant upon exposure to H 2 O 2 for 5 min ( Figure 8D). These results suggest that PlATG6a is involved in the oxidative stress response.

Knockout of PlATG6a Did Not Affect the Oospore Production of P. litchii
To investigate the function of PlATG6a in the sexual stage, we examined the oospore production of WT and ∆platg6a mutants on CJA medium and found no significant difference between the ∆platg6a mutants and WT (Figure 9). In addition, we also found that the oospore size and morphological characteristics of the ∆platg6a mutant were not significantly different from WT. On the basis of these results, we infer that PlATG6a is not required for oospore formation.
Images were taken at 7 dpi. (B) The colony diameters were measured at 7 dpi. Growth inhibition rate (%) was calculated. WT and CK strains were used as controls. (C) Transcriptional analysis of the PlATG6a gene under oxidative stress (5 mM H2O2, for 0, 5, 15, 55 min). Expression levels were normalized using the values at 0 min as '1′. (D) qRT-PCR analysis of P. litchii putative peroxidaseencoding genes in Δplatg6a mutants and WT strain under oxidative stress conditions (5 mM H2O2, for 5 min). Data are mean ± SD (n = 9). Asterisks represent significant differences vs. WT (** p < 0.01) based on t-test. We next assessed the expression level of PlATG6a under oxidative stress, at different time points post exposure to H2O2, following the established method [15,30,31]. The result showed that transcription of PlATG6a was significantly up-regulated at 5 to 55 min after H2O2 treatment ( Figure 8C), implying that this gene plays a role in response to oxidative stress. Furthermore, we examined the expression level of five selected peroxidase-encoding genes [31] in the WT strain and the Δplatg6a mutant. Four out of the five tested genes displayed a higher expression level in the WT strain than that in the mutant upon exposure to H2O2 for 5 min ( Figure 8D). These results suggest that PlATG6a is involved in the oxidative stress response.

Knockout of PlATG6a Did Not Affect the Oospore Production of P. litchii
To investigate the function of PlATG6a in the sexual stage, we examined the oospore production of WT and Δplatg6a mutants on CJA medium and found no significant difference between the Δplatg6a mutants and WT (Figure 9). In addition, we also found that the oospore size and morphological characteristics of the Δplatg6a mutant were not significantly different from WT. On the basis of these results, we infer that PlATG6a is not required for oospore formation. Data are mean ± SD (n = 9). There was no significant difference in oospore number between WT and the Δplatg6a mutant, T32 or T47 (p > 0.5; t-test).

Discussion
As in other eukaryotes, autophagy-related (ATG) genes are conserved and play important roles in growth, development and pathogenicity in filamentous fungi [21]. Their specific roles need to be investigated in different organisms, considering the influence of evolutionary forces on autophagic processes [19]. In this study, we generated deletion mutants of the PlATG6a gene using CRISPR/Cas9 technology, for functional characterization. Our results suggest that PlATG6a is involved in autophagy and contributes to growth, asexual development and tolerance to oxidative stress in P. litchii.
PlAGT6a was up-regulated in zoospores and cysts, and therefore might be critical for asexual development, but not in the infection stage, which is different from the report that PsATG6a in P. sojae was up-regulated during the infection of a host plant [15]. We infer that this different transcriptional profile may reflect the different function of these two Data are mean ± SD (n = 9). There was no significant difference in oospore number between WT and the ∆platg6a mutant, T32 or T47 (p > 0.5; t-test).

Discussion
As in other eukaryotes, autophagy-related (ATG) genes are conserved and play important roles in growth, development and pathogenicity in filamentous fungi [21]. Their specific roles need to be investigated in different organisms, considering the influence of evolutionary forces on autophagic processes [19]. In this study, we generated deletion mutants of the PlATG6a gene using CRISPR/Cas9 technology, for functional characterization. Our results suggest that PlATG6a is involved in autophagy and contributes to growth, asexual development and tolerance to oxidative stress in P. litchii.
PlAGT6a was up-regulated in zoospores and cysts, and therefore might be critical for asexual development, but not in the infection stage, which is different from the report that PsATG6a in P. sojae was up-regulated during the infection of a host plant [15]. We infer that this different transcriptional profile may reflect the different function of these two oomycete ATG6a proteins in growth and plant infection, although they share 84% similarity with each other [15].
The formation of the sporangiophore and its branches is very important for the asexual reproduction of many oomycetes, such as P. litchii, Peronosporaceae species and some Phytophthora species. The sporangiophore and branches determine the production of sporangia and zoospores, which is important for the spread of pathogens from host to host [24]. In this study, we found that knockout of PlATG6a impaired sporangium production. Our results are consistent with previous studies which found that ATG6a is involved in sporangium production in P. sojae and conidia formation in M. oryzae [15,20]. Here, we further revealed that the branches of the sporangiophore were reduced in the ∆platg6a mutants, likely accounting for impairment of sporangium production. However, the mechanism of ATG6a's regulation of sporangium production may be not entirely the same in P. litchii and P. sojae, because there are hardly any sporangiophore branches produced by wild-type P. sojae. The mechanism of sporangiophore formation is not comprehensively known. A previous study reported that PlBZP32 negatively regulated the branch formation of sporangiophores [28]. Here, we reported that PlATG6 is a positive regulator in sporangiophore branch formation. Further study on the relationship between PlATG6a and PlBZP32 might provide a more detailed mechanism of asexual reproduction.
In C. neoformans, deletion of ATG6 affected laccase production, indicating that the Atg6-containing PI3K complex regulates laccase production [19]. It has also been shown that the C. neoformans atg6∆ mutant is sensitive to 10 mM H 2 O 2 treatment [19]. It has not been reported whether the oomycete ATG genes play a role in regulating laccase activity or tolerance to oxidative stress. In this study, we reported for the first time that PlATG6 is involved in P. litchii's response to oxidative stress. However, the deletion of PlATG6a did not affect laccase activity in P. litchii. These results indicate the functional differentiation of ATG6 orthologs in oomycetes and fungi.
M. oryzae ATG6 and P. sojae ATG6a are reported to be required for host plant infection [15,20]. Unlike PsATG6a, PlATG6a is not required for the virulence of P. litchii zoospores, although they share high similarity. Overall, our results display a functional differentiation of ATG6a orthologs in P. sojae and P. litchii during plant infection. When we inoculated litchi leaves with mycelial plugs, the lesion length caused by the ∆platg6a mutants was significantly reduced compared with WT and CK strains. We inferred that the reduced pathogenicity of the ∆platg6a mycelial plugs might be due to fewer sporangia being produced by the mutant. Meanwhile, the functional difference of PlATG6a and PsATG6a during lesion expansion should also be associated with the different transcriptional profiles of these two genes.
In P. litchii, PlATG6b (the ortholog of PsATG6b in P. litchii) shared 22% identity and 34% similarity with PlATG6a. Further study is required for the function of PlATG6b, and for the relationship between PlATG6a and PlATG6b.
This study adds new evidence that the autophagy process is required for the pathogenic development of oomycetous pathogens, besides the limited research on P. infestans and P. sojae that has previously been reported [15,22]. Further studies are needed to reveal how the autophagy process participates in the development and pathogenesis of oomycete pathogens.
In summary, we identified an ATG gene, PlATG6a, in P. litchii and demonstrated that PlATG6a is involved in growth, development, pathogenesis and tolerance to salt and oxidative stress. Furthermore, for the first time we reveal that PlATG6a is a positive regulator of sporangiophore branch formation. This study provides new insight into the mechanism of PlATG6a's involvement in growth, development and oxidative stress in P. litchii.

Strains and Sequence
The P. litchii wild-type strain SHS3 (WT) was isolated and identified in this laboratory. P. litchii WT, CK and ∆platg6a mutants were maintained on carrot juice agar (CJA) medium (juice from 300 g carrot for 1 L medium, 15 g agar/L for solid media) at 25 • C in the dark. The control (CK) strain was a transformant that failed to knockout PlATG6a. The genome sequence and gene annotations of P. litchii were obtained from NCBI (BioProject ID: PRJNA290406) [32], other sequences were obtained from the JGI genome portal (https://genome.jgi.doe.gov/portal/ accessed on 24 January 2022) and previous study [15]. The amino acid sequence alignment was generated and adjusted in BioEdit (version 7.0.9.1).

Transcriptional Level Analysis
The total RNA of P. litchii was extracted using the All-In-One DNA/RNA Mini-preps Kit (Bio Basic, Shanghai, China). The FastKing RT Kit (TIANGEN, Beijing, China) was used for the first-strand cDNA synthesis. The transcriptional levels of PlATG6a were analyzed with quantitative reverse transcription PCR (qRT-PCR) using SYBR ® Premix Ex Taq TM II (TaKaRa, Shiga, Japan). The P. litchii actin gene (PlActin) was used as a loading control [33], and the relative fold change was calculated using the 2 −∆∆CT method [23]. The primers used for these analyses are listed in Supplementary Table S1.

Growth and Development Analysis
To test growth rate, P. litchii WT, CK and ∆platg6a mutants were inoculated on plates (diameter = 9 cm) containing 15 mL CJA medium and cultured at 25 • C in the dark. The colony diameter size was measured and the growth rate was calculated and photographed 5 days after inoculation. Statistical analysis was performed by t-test. The experiments were repeated three times.
For asexual development assays, the methods were described previously [31]. Five 9 mm diameter mycelial plugs were flushed with 2.5 mL double-sterilized water to obtain the sporangia suspension. Then the sporangia were purified using a 100 µm mesh filter. The sporangia were incubated at 16 • C for one hour for release of zoospores. Zoospores were encysted by shaking the suspension for 30 s on an oscillator. Cysts were incubated at 25 • C, 60 rpm for 1 or 2 h for germination. The number of oospores was measured as previously described [33].

CRISPR/Cas9 Gene Editing for PlATG6a
Two sgRNAs were designed and inserted into the sgRNA vector pYF2.3G-Ribo-sgRNA as previously described [34,35]. To generate gene-replacement constructs, 1 kb long upstream/downstream arms of the PlATG6a coding region were inserted into pBluescript II KS vector ( Figure 3A). The pYF2.3G-RibosgRNA (PlATG6a) vector, the hSPCas9 vector pYF2-PsNLS-hSpCas9 and the pBluescript II KS (PlATG6a) vector were co-transformed into protoplasts of P. litchii by PEG-mediated protoplast transformation technology [34]. Preliminary transformants were screened by CJA medium containing 50 µg/mL G418. These transformants were further verified by genomic PCR and subsequent sequencing. These primers are listed in Supplementary Table S1. 4.5. Pathogenicity Assays P. litchii WT, CK and mutants were inoculated on litchi leaves with 6 mm mycelial plugs or 100 zoospores. Then, the inoculated leaves were maintained at 25 • C in the dark. The diameter of the lesions was measured and calculated 48 h after inoculation. The significant differences were analyzed with t-tests. These experiments were repeated three times.

Sensitivity to Various Stress
To investigate the sensitivity of ∆platg6a mutants under different stress conditions, the mycelial plugs (diameter = 9 mm) of ∆platg6a mutants were inoculated in the center of the Plich medium [36]. The Plich media were supplemented with 0.05 mM NaCl or 0.1 mM CaCl 2 . The growth inhibition rate was calculated 7 days after inoculation at 25 • C in the dark. WT and CK strains were used as controls. Growth inhibition rate (%) = (growth diameter on stress-free plates-growth diameter on stress plates)/growth diameter on stress-free plates × 100%.
To analyze the expression levels of PlATG6a under oxidative stress, the WT strain was cultured in liquid Plich medium for 3 days at 25 • C in the dark. Then, the mycelia were immersed in the liquid medium supplemented with 5 mM H 2 O 2 for 0, 5, 15 or 55 min. All samples were harvested and the expressional levels of PlATG6a were evaluated by qRT-PCR.