3.2 RNA sequencing，identification and functional a...
About 4 Gb clean reads(removing sequencing adapters and low-quality bases)were generated for each sample(Table 2).Q20s of all samples were above 96.5%and Q30s were above 92%in both hy4 and Cg01(Table 2).The GC content of each sample was almost 54%in hy4 and 57%in Cg01.Reads of all samples mapped～90% of the reference genomes(Table 2).Among the mapped reads，～91.90%-93.60% mapped to the exons in hy4 samples，while～75.80%-79.70% mapped to the exons in Cg01 samples(Table 2).
Figure 4 Venn diagram of gene annotation in four databases(KOG，Refseq，KEGG，and
Swiss-Prot)of P.polonicum hy4(A)and C.gloeosporioides Cg01(B)
Table 2 RNA sequencing information for the two HupA-producing endophytes P.polonicum hy4 and C.gloeosporioides Cg01 in the control group and induced group
Note：hy4_*and Cg_*represent the control groups of hy4 and Cg01；hy4_in_*and Cg_in_*represent the induced groups of hy4 and Cg01.Each experiment was performed in triplicate.
Distinctive expression patterns of genes between the control and induced group in hy4 and Cg01 provide an opportunity to find important genes that were functionally responsive to the addition of H.serrata extracts and might correlate with Hup A biosynthesis.Among the 11，696 genes in hy4，3，784(32.4%)were differentially expressed after induction(Q-value≤0.05，|log2(fold_change)|≥1)，with 1，894(16.2%)up-regulated(Figure 5).In Cg01，3，813 genes(21.53%)were differentially expressed(Q-value≤0.05，|log2(fold_change)|≥1)，with 2，263(19.0%)upregulated(Figure 5).
Figure 5 Volcano plots of differentially expressed genes in two endophytic fungi(A)P.polonicum hy4；(B)C.gloeosporioides Cg01
Volcano plots visualize the DEGs between the two different groups.Q-values≤0.05 and|log2(fold_change)|≥1 were used as a threshold to determine the significance of DEGs.Red dots：up-regulated genes；Blue dots：down-regulated genes；Gray dots：genes with no significant difference.
DEGs in hy4 and Cg01 belonged to different functional pathways，including transporters(160/144)，transcription factors(93/79)，tryptophan metabolism(78/63)，etc.However，there was no shared pathway between hy4 and Cg01 with Q-value≤0.05.KEGG enrichment pathways were“RNA polymerase”and“Starch and sucrose metabolism”in hy4，while for Cg01 it was“Citrate cycle”.With P-value≤0.05，the shared enrichment pathways“Glycolysis/Gluconeogenesis”and“Transcription factors” were most affected by the addition of H.serrata extracts.Because Hup A-producing ability was improved after induction in both hy4 and Cg01，the shared enrichment pathways with similar expression pattern were the focuses.With Q-value≤1，the shared enrichment pathways with similar expression pattern were Glycolysis/“Gluconeogenesis”，“Tryptophan metabolism”，“Methionine metabolism”，and“Glutamate metabolism”(Figure 6).(https://www.daowen.com)
The GO consortium provides a standardized and hierarchical vocabulary(GO terms)to describe the function of gene products and classifies genes into functional categories.Of the significantly changed genes，30 significantly enriched GO terms were obtained，divided into three main categories(biological processes，cellular components，and molecular functions)(Figure 7).The most enriched GO pathways(Q-value≤0.05)included 16 pathways in hy4 and 3 in Cg01(Figure 7).The shared enrichment pathways between hy4 and Cg01 were preribosome，large subunit precursor，and 90S preribosome.
Figure 6 Scatter plot of top 20 enriched KEGG pathways for genes with significant expression levels in two endophytic fungi(A)P.polonicum hy4(B)C.gloeosporioides Cg01
A high Q-value is represented by blue and a low Q-value is represented by red.Bubble size indicates DEG number(increases with DEG number).
Figure 7 Bar chart of top 30 GO terms for genes with significantly enhanced expression in two endophytic fungi(A)P.polonicum hy4；(B)C.gloeosporioides Cg01
Symbol*indicates enrichment was significant(Qvalue＜0.05).The number after the GO term is the number of genes in the enrichment GO terms.
We included for comprehensive consideration the DEGs in GO and KEGG pathways with similar expression pattern in hy4 and Cg01.The DEGs were mostly involved in carbon metabolism(Glycolysis/Gluconeogenesis)，nitrogen metabolism(Methionine metabolism，Tryptophan metabolism，Glutamate metabolism)and in the nucleolus，ribosome，and rRNA processing(Figure 8).Most of the shared DEGs in“Glycolysis/Gluconeogenesis”were upregulated，while most shared DEGs in amino acid metabolism(Methionine metabolism，Tryptophan metabolism，Glutamate metabolism)were downregulated(Figure 8).
Figure 8 Model illustrating the KEGG pathways and GO function clusters of the potential DEGs involved in HupA biosynthesis
The number aboveeach horizontal arrow is the ECnumber of theenzyme catalyzing this reaction，while red represents upregulation，green represents downregulation，and blue represents genes that both up-and down-regulate.