Cmfhp Gene Mediates Fruiting Body Development and Carotenoid Production in Cordyceps militaris.

Cordyceps militaris fruiting bodies contain a variety of bioactive components that are beneficial to the human body. However, the low yield of fruiting bodies and the low carotenoid content in C. militaris have seriously hindered the development of the C. militaris industry. To elucidate the developmental mechanism of the fruiting bodies of C. militaris and the biosynthesis mechanism of carotenoids, the function of the flavohemoprotein-like Cmfhp gene of C. militaris was identified for the first time. The Cmfhp gene was knocked out by the split-marker method, and the targeted gene deletion mutant ΔCmfhp was obtained. An increased nitric oxide (NO) content, no fruiting body production, decreased carotenoid content, and reduced conidial production were found in the mutant ΔCmfhp. These characteristics were restored when the Cmfhp gene expression cassette was complemented into the ΔCmfhp strain by the Agrobacterium tumefaciens-mediated transformation method. Nonetheless, the Cmfhp gene had no significant effect on the mycelial growth rate of C. militaris. These results indicated that the Cmfhp gene regulated the biosynthesis of NO and carotenoids, the development of fruiting bodies, and the formation of conidia. These findings potentially pave the way to reveal the developmental mechanism of fruiting bodies and the biosynthesis mechanism of carotenoids in C. militaris.


Introduction
Cordyceps militaris is a well-known medicinal and edible filamentous fungus. It has been widely used in many countries because it contains many kinds of bioactive components (such as cordycepin, cordycepic acid, polysaccharides, carotenoids, ergosterol, etc.) [1][2][3][4]. These bioactive components endow C. militaris with anticancer, antitumor, anti-inflammatory, and antioxidant activities [5][6][7]. In recent years, novel carotenoids with good water solubility and pentostatin with anticancer activity have been found in C. militaris, and these findings have increased the market demand for C. militaris every year [8,9].
Natural carotenoids play an important role in human health and food coloring. It was suggested that the carotenoid content should be considered as the quality standard of the commercial products of C. militaris [10]. C. militaris, a potential natural carotenoid resource, cannot meet the market demand because of its low carotenoid content [11,12]. A good way to improve the carotenoid content of The deletion of the Cmfhp gene was further confirmed by quantitative real-time PCR (qRT-PCR) using the tef1 gene (GenBank: DQ070019) as the internal control gene [25]. Primers tef1-F and tef1-R (for detecting the tef1 gene) and primers Qfhp-F and Qfhp-R (for detecting the Cmfhp gene) are listed in Table S1. All qRT-PCR was carried out according to previously described methods [26]. The relative expression level of the Cmfhp gene was calculated relative to tef1 expression using the 2 −∆∆CT method [27].

Complementation of the Cmfhp Disruption Mutant
The A. tumefaciens AGL1-pCAMBIA0390-Ben-Comfhp and conidia of the mutant ∆Cmfhp were co-cultured to achieve complementation of the Cmfhp gene by the A. tumefaciens-mediated transformation (ATMT) method [28]. Colonies that could grow on resistant PDA containing 3 µg/mL of benomyl were considered to be putative transformants. The successful complementary transformants (∆Cmfhp-c) were verified by PCR and qRT-PCR.

Determination of the NO Content
The NO content in C. militaris was determined using an NO assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. One gram of fresh C. militaris mycelia was ground in 5 mL of 40 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.2) to assess the NO content. The homogenate was centrifuged at 14,000× g for 10 min. The supernatant was used to measure NO [29]. Three biological replicate experiments were performed on each strain of C. militaris.

Cultivation of C. militaris Fruiting Bodies
The fruiting bodies of all C. militaris strains (CM10, ∆Cmfhp, and ∆Cmfhp-c) were cultured on rice medium according to a previously described method [14].

Determination of Carotenoid Content
All C. militaris strains (CM10, ∆Cmfhp, and ∆Cmfhp-c) were cultured on PDA for 3 weeks under dark conditions and then 1 week under light conditions. The C. militaris mycelia were collected and vacuum freeze-dried. The dried mycelia were used for the determination of carotenoid content according to previously reported methods [10,12].

Growth Rate and Conidial Production
The strains of C. militaris (CM10, ∆Cmfhp, and ∆Cmfhp-c) were inoculated on PDA at 25 • C for 3 weeks before use. A 5-mm disk was punched with a sterilized cutter from the prepared PDA inoculum and transferred to a fresh PDA plate. PDA plates with C. militaris inoculum were cultured in the dark at 25 • C. The growth rate was determined by measuring the colony diameter after 3 weeks of incubation [12]. Then, the colonies on PDA medium that had been cultured for 3 weeks were used to determine the production of conidia. Mycelia were scraped from the PDA plates and resuspended in 10 mL of a Tween 80 solution (20%, w/v). After filtration, the conidial suspensions were counted using a hemocytometer under a microscope [18,30].

Statistical Analysis
All experiments were carried out in triplicate. Data were analyzed by SPSS 22.0 software (SPSS Inc., Chicago, IL, USA). The values are shown as the mean ± standard error. p-values less than 0.05 were considered significant.

Disruption and Complementation of the Cmfhp Gene
Using the plasmid pCAMBIA0390-Bar-KOfhp as a DNA template, the 5 split-marker fragment and the 3 split-marker fragment were prepared by PCR amplification (Figure 1a,b). C. militaris protoplasts were prepared from mycelia ( Figure 1c). Then, 5 split-marker fragment and the 3 split-marker fragment were co-transformed into C. militaris protoplasts. The results of PCR analysis showed that the bar gene was successfully integrated into the C. militaris genome and that the Cmfhp gene was knocked out (Figure 1d). The results of hybridization with the bar probe suggested that there were three hybridization bands in the PCR-positive mutant of lane 19, one hybridization band in the PCR-positive mutant of lane 20, and no hybridization band in wild-type C. militaris (Figure 1e).

Disruption and Complementation of the Cmfhp Gene
Using the plasmid pCAMBIA0390-Bar-KOfhp as a DNA template, the 5′ split-marker fragment and the 3′ split-marker fragment were prepared by PCR amplification (Figure 1a,b). C. militaris protoplasts were prepared from mycelia ( Figure 1c). Then, 5′ split-marker fragment and the 3′ splitmarker fragment were co-transformed into C. militaris protoplasts. The results of PCR analysis showed that the bar gene was successfully integrated into the C. militaris genome and that the Cmfhp gene was knocked out (Figure 1d  The mutant corresponding to lane 20 was used for qRT-PCR analysis. The qRT-PCR results demonstrated that the expression of the Cmfhp gene was not detected in the mutant ΔCmfhp ( Figure  2). Based on these results, the Cmfhp gene was successfully knocked out, and the ΔCmfhp strain was used for subsequent experiments. The mutant corresponding to lane 20 was used for qRT-PCR analysis. The qRT-PCR results demonstrated that the expression of the Cmfhp gene was not detected in the mutant ∆Cmfhp ( Figure 2). Based on these results, the Cmfhp gene was successfully knocked out, and the ∆Cmfhp strain was used for subsequent experiments.

Effect of the Cmfhp Gene on the NO Content in C. militaris
Compared with wild-type C. militaris CM10, C. militaris ΔCmfhp contained more NO, which might be due to the deletion of the Cmfhp gene. Moreover, the NO content in the complementary strain ΔCmfhp-c was restored to the levels in wild-type C. militaris CM10 (Figure 4). Therefore, the expression product of the Cmfhp gene could metabolize NO in C. militaris and reduce the content of NO in C. militaris, and these effects were consistent with the previously reported function of flavohemoprotein [31].

Effect of the Cmfhp Gene on the NO Content in C. militaris
Compared with wild-type C. militaris CM10, C. militaris ΔCmfhp contained more NO, which might be due to the deletion of the Cmfhp gene. Moreover, the NO content in the complementary strain ΔCmfhp-c was restored to the levels in wild-type C. militaris CM10 (Figure 4). Therefore, the expression product of the Cmfhp gene could metabolize NO in C. militaris and reduce the content of NO in C. militaris, and these effects were consistent with the previously reported function of flavohemoprotein [31].

Effect of the Cmfhp Gene on the NO Content in C. militaris
Compared with wild-type C. militaris CM10, C. militaris ∆Cmfhp contained more NO, which might be due to the deletion of the Cmfhp gene. Moreover, the NO content in the complementary strain ∆Cmfhp-c was restored to the levels in wild-type C. militaris CM10 (Figure 4). Therefore, the expression product of the Cmfhp gene could metabolize NO in C. militaris and reduce the content of NO in C. militaris, and these effects were consistent with the previously reported function of flavohemoprotein [31].

Effect of the Cmfhp Gene on the Development of C. militaris Fruiting Bodies
The fruiting bodies of wild-type C. militaris CM10 cultured on rice medium were irregular lumps in shape and were orange in color (Figure 5a). However, C. militaris ∆Cmfhp cultured on rice medium did not produce fruiting bodies (Figure 5b). When the Cmfhp gene was complemented to the mutant ∆Cmfhp, the complementary strain ∆Cmfhp-c had a restored ability to produce fruiting bodies.
Moreover, the fruiting bodies of the complementary strain ∆Cmfhp-c were still irregular lumps in shape and were orange in color (Figure 5c). These results suggested that the Cmfhp gene was involved in the development of C. militaris fruiting bodies.

Effect of the Cmfhp Gene on the Development of C. militaris Fruiting Bodies
The fruiting bodies of wild-type C. militaris CM10 cultured on rice medium were irregular lumps in shape and were orange in color (Figure 5a). However, C. militaris ΔCmfhp cultured on rice medium did not produce fruiting bodies (Figure 5b). When the Cmfhp gene was complemented to the mutant ΔCmfhp, the complementary strain ΔCmfhp-c had a restored ability to produce fruiting bodies. Moreover, the fruiting bodies of the complementary strain ΔCmfhp-c were still irregular lumps in shape and were orange in color (Figure 5c). These results suggested that the Cmfhp gene was involved in the development of C. militaris fruiting bodies.

Effect of the Cmfhp Gene on the Development of C. militaris Fruiting Bodies
The fruiting bodies of wild-type C. militaris CM10 cultured on rice medium were irregular lumps in shape and were orange in color (Figure 5a). However, C. militaris ΔCmfhp cultured on rice medium did not produce fruiting bodies (Figure 5b). When the Cmfhp gene was complemented to the mutant ΔCmfhp, the complementary strain ΔCmfhp-c had a restored ability to produce fruiting bodies. Moreover, the fruiting bodies of the complementary strain ΔCmfhp-c were still irregular lumps in shape and were orange in color (Figure 5c). These results suggested that the Cmfhp gene was involved in the development of C. militaris fruiting bodies.

Effect of the Cmfhp Gene on the Carotenoid Content of C. militaris
The C. militaris strains CM10, ∆Cmfhp, and ∆Cmfhp-c were all cultured on PDA medium (Figure 5d-f). The C. militaris strains CM10 and ∆Cmfhp-c were all orange in color; however, the color of the C. militaris strain ∆Cmfhp was light yellow. These results implied that the Cmfhp gene regulated the production of C. militaris pigments. In addition, the analysis of carotenoid content showed that the carotenoid content of C. militaris CM10 was not significantly different from that of C. militaris ∆Cmfhp-c but was significantly higher than that of C. militaris ∆Cmfhp (Figure 5g). Therefore, we believe that the Cmfhp gene regulated the production of carotenoids in C. militaris.

Effect of the Cmfhp Gene on the Growth Rate and Conidial Production of C. militaris
The analysis results of the mycelial growth rate showed that there was no significant difference in the growth rates of the C. militaris strains CM10, ∆Cmfhp, and ∆Cmfhp-c (Figure 5h). This indicated that the Cmfhp gene had no significant effect on the growth of C. militaris. However, the conidial production of the mutant ∆Cmfhp was significantly lower than that of the wild-type C. militaris, and there was no significant difference in the conidial production between the wild-type strain CM10 and the complementary strain ∆Cmfhp-c (Figure 5i). These results demonstrated that the Cmfhp gene significantly affected the conidial production of C. militaris.

Discussion
Light is a necessary condition for the production of C. militaris fruiting bodies and pigments [18]. The Cmfhp gene of C. militaris was significantly upregulated after being treated with light. In the present study, the function of the Cmfhp gene in C. militaris was studied for the first time by gene knockout and gene complementation. We found that the Cmfhp gene not only affected the formation of C. militaris fruiting bodies and conidia but also regulated the production of NO and carotenoids. However, the Cmfhp gene had no significant effect on the growth rate of C. militaris mycelia.
Flavohemoprotein, a NO dioxygenase, is capable of oxidizing NO to nitrate using oxygen [22]. This enzymatic conversion thus protects the cell from toxic NO and from other damaging NO-derived reactive nitrogen species [22]. The deletion of the Cmfhp gene led to an accumulation of more NO in the ∆Cmfhp mutant, which might be due to the absence of the flavohemoprotein (encoded by the Cmfhp gene) that could catalyze the oxidation of NO to nitrate. In addition, NO produced at sufficient levels directly or indirectly damages critical cell processes [32]. Hence, excessive NO might be toxic to the development and metabolism of C. militaris.
The consumer's demand for C. militaris is mainly for its fruiting bodies. However, the developmental mechanism of C. militaris fruiting bodies is poorly understood. Although C. militaris is a heterothallic ascomycetous fungus, it can still produce fruiting bodies without an opposite mating-type partner [33]. Previous studies have also shown that the mating-type is not the decisive factor for the production of C. militaris fruiting bodies [34,35]. Therefore, it is necessary to study the key genes involved in the formation of C. militaris fruiting bodies. In this study, the wild-type C. militaris CM10 with a single mating-type gene (MAT 1-1) can stably produce irregular lumpy fruiting bodies. The phenotypic analysis of the fruiting bodies of C. militaris strains (CM10, ∆Cmfhp, and ∆Cmfhp-c) revealed that the Cmfhp gene was a key gene affecting the formation of C. militaris fruiting bodies. It has been previously reported that NO regulates the formation of fungal fruiting bodies [21]. Therefore, we believe that the loss of the ability to produce fruiting bodies of the ∆Cmfhp strain may be due to its high NO content.
Carotenoids are secondary metabolites produced by C. militaris cultured under the light. However, the biosynthetic pathway of C. militaris carotenoids is still unknown, and there are few reports on the genes involved in the biosynthesis of C. militaris carotenoids. A putative carotenoid biosynthetic pathway for C. militaris was proposed, but 11 genes involved in this putative pathway were not significantly differentially expressed between the C. militaris strain 498 and the C. militaris strain 505 after light irradiation [13]. Although three types of geranylgeranyl diphosphate synthase genes in C. militaris were cloned, their functions have yet to be identified [36]. It is noteworthy that the other two key enzymes (phytoene synthetase and phytoene dehydrogenase) were not found in the genome of C. militaris [13,18]. The induction of carotenoids was completely different between C. militaris and Neurospora crassa [13,18]. Therefore, it is a great challenge to characterize the carotenoid biosynthetic pathway in C. militaris. In this study, the Cmfhp gene was identified to have a significant effect on the biosynthesis of carotenoids in C. militaris. The carotenoid content of the ∆Cmfhp strain was lower than that of wild-type C. militaris CM10, while the NO content of the ∆Cmfhp strain was significantly higher than that of wild-type C. militaris CM10. It has been reported that NO could downregulate the synthesis of carotenoids in Chlamydomonas reinhardtii [37]. Therefore, we believed that the excessive NO in the ∆Cmfhp strain inhibited the biosynthesis of carotenoids.
NO inhibited the mycelial growth of Aspergillus niger, Monilinia fructicola, Penicillium italicum, and N. crassa [21,38]. In the present study, although the NO content in the ∆Cmfhp strain was higher than that in wild-type C. militaris CM10, there was no significant difference in their growth rate. This might be due to the different tolerance thresholds of different fungi to NO, and the NO content in the ∆Cmfhp strain might not reach the level that inhibits the growth of C. militaris mycelia. It has also been reported that NO inhibited the conidial production of N. crassa [21,39]. In this study, the conidial production in C. militaris (CM10, ∆Cmfhp, and ∆Cmfhp-c) was negatively correlated with the content of NO. This suggests that the conidial production of C. militaris may be inhibited by NO.
Flavohemoprotein could detoxify NO in A. fumigatus [31]. However, the detoxification mechanism of NO in C. militaris is still unknown. Therefore, the following aspects will need to be studied: (1) elucidating the formation mechanism of NO in C. militaris; (2) revealing the expression mechanism of the Cmfhp gene in C. militaris; (3) clarifying the NO detoxification mechanism of the Cmfhp gene; (4) elucidating the regulation mechanism of NO on the development of fruiting bodies and the metabolism of bioactive ingredients in C. militaris. The identification of the function of the Cmfhp gene in this study will also help to reveal the developmental mechanism of fruiting bodies and the biosynthesis mechanism of carotenoids in C. militaris.

Conclusions
The function of the Cmfhp gene in C. militaris was identified by gene knockout and gene complementation for the first time, and the target gene deletion mutant ∆Cmfhp and the target gene complementary strain ∆Cmfhp-c were obtained. The deletion of the Cmfhp gene resulted in an increase in NO content, the loss of the ability to produce fruiting bodies, a decrease in the carotenoid content, and a reduction in conidial production in the ∆Cmfhp mutant. However, the deletion of the Cmfhp gene had no significant effect on the mycelial growth rate of C. militaris. The identification of the function of the Cmfhp gene will be helpful to reveal the developmental mechanism of C. militaris fruiting bodies and the metabolic regulation mechanism of carotenoids in C. militaris.

Conflicts of Interest:
The authors declare no conflict of interest.