Proteome and Transcriptome Reveal Involvement of Heat Shock Proteins and Indoleacetic Acid Metabolism Process in Lentinula Edodes Thermotolerance.

BACKGROUND/AIMS: Heat stress could cause huge losses for Lentinula edodes in China and other Asian cultivation areas. Yet our understanding of mechanism how to defend to heat stress is incomplete. METHODS: Using heat-tolerant and heat-sensitive strains of L. edodes, we reported a combined proteome and transcriptome analysis of L. edodes response to 40 °C heat stress for 24 h. Meanwhile, the effect of LeDnaJ on the thermotolerance and IAA (indoleacetic acid) biosynthesis in L. edodes was analyzed via the over-expression method. RESULTS: The proteome results revealed that HSPs (heat shock proteins) such as Hsp40 (DnaJ), Hsp70, Hsp90 and key enzymes involved in tryptophan and IAA metabolism process LeTrpE, LeTrpD, LeTam-1, LeYUCCA were more highly expressed in S606 than in YS3357, demonstrating that HSPs and tryptophan as well as IAA metabolism pathway should play an important role in thermotolerance. Over-expression of LeDnaJ gene in S606 strains showed better tolerance to heat stress. It was also documented that intracellular IAA accumulation of S606 (8-fold up) was more than YS3357 (2-fold up), and exogenous IAA enhanced L. edodes tolerance to heat stress. CONCLUSION: Our data support the interest of LeTrpE, LeDnaJ, tryptophan and IAA could play a pivotal role in enhancing organism thermotolerance.

Genetic dissection of fruiting body-related traits using quantitative trait loci mapping in Lentinula edodes.

To provide a better understanding of the genetic architecture of fruiting body formation of Lentinula edodes, quantitative trait loci (QTLs) mapping was employed to uncover the loci underlying seven fruiting body-related traits (FBRTs). An improved L. edodes genetic linkage map, comprising 572 markers on 12 linkage groups with a total map length of 983.7 cM, was constructed by integrating 82 genomic sequence-based insertion-deletion (InDel) markers into a previously published map. We then detected a total of 62 QTLs for seven target traits across two segregating testcross populations, with individual QTLs contributing 5.5 %-30.2 % of the phenotypic variation. Fifty-three out of the 62 QTLs were clustered in six QTL hotspots, suggesting the existence of main genomic regions regulating the morphological characteristics of fruiting bodies in L. edodes. A stable QTL hotspot on MLG2, containing QTLs for all investigated traits, was identified in both testcross populations. QTLs for related traits were frequently co-located on the linkage groups, demonstrating the genetic basis for phenotypic correlation of traits. Meta-QTL (mQTL) analysis was performed and identified 16 mQTLs with refined positions and narrow confidence intervals (CIs). Nine genes, including those encoding MAP kinase, blue-light photoreceptor, riboflavin-aldehyde-forming enzyme and cyclopropane-fatty-acyl-phospholipid synthase, and cytochrome P450s, were likely to be candidate genes controlling the shape of fruiting bodies. The study has improved our understanding of the genetic architecture of fruiting body formation in L. edodes. To our knowledge, this is the first genome-wide QTL detection of FBRTs in L. edodes. The improved genetic map, InDel markers and QTL hotspot regions revealed here will assist considerably in the conduct of future genetic and breeding studies of L. edodes.

Evaluating genetic diversity and constructing core collections of Chinese Lentinula edodes cultivars using ISSR and SRAP markers.

Genetic diversity among 89 Chinese Lentinula edodes cultivars was analyzed by inter-simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) markers. A 123 out of 126 ISSR loci (97.62%) and 108 out of 129 SRAP loci (83.73%) were polymorphic between two or more strains. A dendrogram constructed by cluster analysis based on the ISSR and SRAP markers separated the L. edodes strains into two major groups, of which group B was further divided into five subgroups. Clustering results also showed a positive correlation with the main agronomic traits of the strains, and that strains with similar traits clustered together into the same groups or subgroups in most cases. The average coefficient of pairwise genetic similarity was 0.820 (range: 0.576-0.988). Compared to the wild strains, Chinese L. edodes cultivars indicated a lower level of genetic diversity. Two preliminary core collections of L. edodes, Core1 and Core2, were established based on the ISSR and SRAP data, respectively. Core1 was constructed by the advanced M (maximization) strategy using the PowerCore version 1.0 software and contained 21 strains, whereas Core2 was created by the allele preferred sampling strategy using the cluster method and contained 18 strains. Both core collections were highly representative of the genetic diversity of the original germplasm, as confirmed by the values of Na (observed number of alleles), Ne (effective number of alleles), H (Nei's gene diversity) and I (Shannon's information index), as well as results of principal coordinate analysis. The loci retention ratio of Core1 (99.61%) was higher than that of Core2 (97.65%). Moreover, Core1 contained strains with more types of agronomic traits than those in Core2. This study builds the basis for further effective protection, management and use of L. edodes germplasm resource.

Efficiency of RAPD and ISSR markers in differentiation of homo- and heterokaryotic protoclones of Agaricus bisporus.

Morphologically, nine different slow-growing protoclones were screened from regenerated protoplast of heterokaryotic Agaricus bisporus. The present study is the first report of fingerprinting on differentiating homo- and heterokaryotic protoclones using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Among 80 primers tested, the seven ISSR and seven RAPD primers selected for the analysis generated a total of 94 ISSR and 52 RAPD fragments, respectively. ISSR fingerprinting detected more polymorphic loci (38.29%) than RAPD fingerprinting (34.61%). Principal coordinate analysis (PCA) was employed to evaluate the resolving power of the markers to differentiate protoclones. The mean polymorphism information content (PIC) for each of these marker systems (i.e., 0.787 for RAPD and 0.916 for ISSR, respectively) suggests that the ISSR marker system was more effective in determining polymorphisms. The dendrograms constructed using RAPD, ISSR, and integrated RAPD, and ISSR marker systems were highly correlated with each other as revealed by the high Mantel correlation (r = 0.98). Pair-wise similarity index values ranged from 0.64 to 0.95 (RAPD), 0.67 to 0.98 (ISSR), and 0.67 to 0.98 (RAPD and ISSR), and mean similarity index values of 0.82, 0.81, and 0.84 for RAPD, ISSR, and combined data, respectively, were obtained. As there was a good correspondence between RAPD and ISSR similarity matrices, ISSR may be used as an alternative to replace RAPD in the genetic diversity assessment and accurate differentiation of homo- and heterokaryotic protoclones of A. bisporus.

Applying target region amplification polymorphism markers for analyzing genetic diversity of Lentinula edodes in China.

The target region amplification polymorphism (TRAP) technique was utilized for assessing the genetic diversity of 55 wild strains and one cultivated strain of Lentinula edodes in China. From these strains, 932 DNA fragments were amplified using 12 primer combinations, 929 fragments (99.68%) of which were polymorphic between two or more strains. The average coefficient of pairwise genetic similarity was 0.696, within a range from 0.503 to 0.947. Cluster analysis and principal coordinate analysis separated the tested strains of L. edodes into two major groups. Group A was further divided into seven subgroups. In most cases, the strains from the same or adjoining regions could be preferentially clustered into small groups. The results from the average genetic similarity and the weighted average value of Shannon's Information Index among the tested strains of L. edodes from the same region revealed a vast genetic diversity in the natural germplasm found in China. Compared with the L. edodes strains from other regions, those found on the Yunnan Plateau, in the Hengduanshan Mountains, in Taiwan, South China, and Northeast China showed greater genetic diversity. The results of the present study indicated that the wild strains of L. edodes in China possessed abundant genetic variation, and the genetic relationships among them were highly associated with the geographic distribution. This is the first report demonstrating that TRAP markers were powerful for analyzing the genetic diversity of L. edodes, and the study lays the foundation for a further application of this remarkable technique to other fungi.

Remediation and Mechanisms of Cadmium Biosorption by a Cadmium-Binding Protein from Lentinula edodes.

Cadmium bioremediation with metal-binding proteins is primarily conducted using metallothioneins (MTs). However, in the present study, we investigated a non-MT cadmium-binding protein from Lentinula edodes (LECBP) as a remediation tool for cadmium biosorption in Escherichia coli. The results indicated that the expression of LECBP significantly enhanced the cadmium biosorption capacity of transgenic E. coli. The secondary structure and conformation of LECBP were changed after binding with cadmium as evidenced by circular dichroism and fluorescence spectroscopy. The results of Fourier transform infrared spectroscopy indicated that carboxyl oxygen and amino nitrogen atoms were involved in the interaction between LECBP and cadmium. The results further demonstrated that glutamic acid and histidine residues are the potential binding sites. Our results have thus provided new insights into cadmium bioremediation in an aquatic environment.

Purification and Characterization of a Cadmium-Binding Protein from Lentinula edodes.

Many organisms possess the ability to produce metal-binding proteins to absorb cadmium. Metallothioneins, an important family of cysteine-rich metal-binding proteins, have been isolated and well characterized. However, Lentinula edodes may have a different type of cadmium-binding protein that contains fewer cysteine residues. In the present study, we purified a cadmium-binding protein from L. edodes (LECBP) by gel filtration and anion exchange chromatography and then identified LECBP by LC-MS/MS. We found LECBP to be a novel cadmium-binding protein, which contained 220 amino acid residues but no cysteine residue. LECBP had a high binding affinity for Cd(II) with a Kd value of 97.3 µM. The percentages of α-helix, ß-sheet, ß-turn, and random coil in LECBP were 15.7%, 39.4%, 8.0%, and 37.1%, respectively. In addition, high temperatures and an acidic environment influenced the conformation of LECBP. Our results will thus provide a new perspective to understand the mechanism of cadmium accumulation in L. edodes.

Fungi-Enabled Synthesis of Ultrahigh-Surface-Area Porous Carbon.

The growth of white-rot fungi is related to the superior infiltrability and biodegradability of hyphae on a lignocellulosic substrate. The superior biodegradability of fungi toward plant substrates affords tailored microstructures, which benefits subsequently high efficient carbonization and chemical activation. Here, the mechanism underlying the direct growth of mushrooms toward the lignocellulosic substrate is elucidated and a fungi-enabled method for the preparation of porous carbons with ultrahigh specific surface area (3439 m2 g-1 ) is developed. Such porous carbons could have potential applications in energy storage, environment treatment, and electrocatalysis. The present study reveals a novel pore formation mechanism in root-colonizing fungi and anticipates a valuable function for fungi in developing the useful porous carbons with a high specific surface area.

A novel cysteine desulfurase influencing organosulfur compounds in Lentinula edodes.

Organosulfur compounds are the basis for the unique aroma of Lentinula edodes, and cysteine sulfoxide lyase (C-S lyase) is the key enzyme in this trait. The enzyme from Alliium sativum has been crystallized and well-characterized; however, there have been no reports of the characterization of fungi C-S lyase at the molecular level. We identified a L. edodes C-S lyase (Lecsl), cloned a gene of Csl encoded Lecsl and then combined modeling, simulations, and experiments to understand the molecular basis of the function of Lecsl. Our analysis revealed Lecsl to be a novel cysteine desulfurase and not a type of cysteine sulfoxide lyase. The pyridoxal-5-phosphate (PLP) molecule bonded tightly to Lecsl to form a Lecsl-PLP complex. Moreover, the Lecsl had one active center that served to bind two kinds of substrates, S-methyl-L-cysteine sulfoxide and L-cysteine, and had both cysteine sulfoxide lyase and cysteine desulfurase activity. We found that the amino acid residue Asn393 was essential for the catalytic activity of Lecsl and that the gene Csl encoded a novel cysteine desulfurase to influence organosulfur compounds in L. edodes. Our results provide a new insight into understanding the formation of the unique aroma of L. edodes.

[Genetic analysis of isozyme loci of intraspecific hybrid in Auricularia auricula].

Monokaryon strains H2 and J3 were respectively developed from the cultivated strains He-1 and Ju-1 of Auricularia auricula through protoplasted monokaryon technique. Dicaryon strain H2J3 was bred through crossbreeding of H2 and J3 as parents. Fifty-two F1 monokaryon strains were obtained from the fruitbodies of dicaryon H2J3 of A. auricula by the single-spore isolation and culture. All the tested strains, parent monokaryon strains (H2,J3) and fifty-two F1 monokaryon strains, were cultured in liquid Complete Yeast Medium(CYM) for twenty days, and the mycelia of tested strains were respectively analysed by polyacrylamide gel electrophoresis. Bian Y B et al. have reported the results of isozyme zymogram polymorphisms of cultured strains including He-1 and Ju-1, and the specific expression of esterase genes on the different stages of mycelial development in A. auricula. The same named methods of isozyme loci and alleles suggested by Gottlieb (1973) were used in this study. The relative fitness of allele segregation and theoretical expected ratio was examined by the contingency chi 2 test. The linkage relationship was assessed according to the method suggested by Rudin and Ekberg (1978). The isozyme analysis of tested strains showed that the isozymes of esterase (EST), malate dehydrogenase(MDH) and formate dehydrogenase(FDH) of A. auricula were respectively controlled by 7, 5 and 4 polymorphic loci. The isozyme bands which were controlled by EST-3, EST-4, MDH-3 and MDH-4 loci were stably expressed in the parental strains and most tested F1 monokaryon strains. Genetic analysis was impossible for these four loci. The relative fitness between allele observed value and theoretical expected value was further examined by means of contingency chi 2 at 1% level, the result indicated that significant difference existed from the chi 2 value of MDH-5 and FDH-1 in 7 polymorphic loci of EST, MDH and FDH, and denied the hypothesis in which the isozyme bands controlled by MDH-5 and FDH-1 loci were presumed to be allozyme bands. The allele observed values (100:SA) of EST-7, MDH-1, MDH-5, FDH-1, FDH-2 and FDH-4 were significant deviation of theoretical expected ratio (1:1), which showed that these variations were not due to the allele expressions. The significant difference was not be showed at the 5% level from the chi 2 values of EST-1, EST-2, EST-5, EST-6 and MDH-2, this result indicated the allele segregation was corresponding to the theoretical expected ratio at these 5 loci, these 5 loci were allozyme loci which was corresponding to Mendel's law of segregation. A total of 10 possible pair combinations from the 5 allozyme loci were tested. The numbers of monokaryon strains with parental type zymogram or recombinant type zymogram were counted up, and the chi 2I, chi 2II and chi 2L were calculated for examining the linkage relationships of these allozyme loci, the chi 2L value attained to significant difference at 1% level between EST-5 and EST-6 loci, it indicated that the linkage relationship existed between the two loci, non-linkage relationships existed between the other pairing loci of 5 allozyme loci.

Constructing a new integrated genetic linkage map and mapping quantitative trait loci for vegetative mycelium growth rate in Lentinula edodes.

The most saturated linkage map for Lentinula edodes to date was constructed based on a monokaryotic population of 146 single spore isolates (SSIs) using sequence-related amplified polymorphism (SRAP), target region amplification polymorphism (TRAP), insertion-deletion (InDel) markers, and the mating-type loci. Five hundred and twenty-four markers were located on 13 linkage groups (LGs). The map spanned a total length of 1006.1 cM, with an average marker spacing of 2.0 cM. Quantitative trait loci (QTLs) mapping was utilized to uncover the loci regulating and controlling the vegetative mycelium growth rate on various synthetic media, and complex medium for commercial cultivation of L. edodes. Two and 13 putative QTLs, identified respectively in the monokaryotic population and two testcross dikaryotic populations, were mapped on seven different LGs. Several vegetative mycelium growth rate-related QTLs uncovered here were clustered on LG4 (Qmgr1, Qdgr1, Qdgr2 and Qdgr9) and LG6 (Qdgr3, Qdgr4 and Qdgr5), implying the presence of main genomic areas responsible for growth rate regulation and control. The QTL hotspot region on LG4 was found to be in close proximity to the region containing the mating-type A (MAT-A) locus. Moreover, Qdgr2 on LG4 was detected on different media, contributing 8.07 %-23.71 % of the phenotypic variation. The present study provides essential information for QTL mapping and marker-assisted selection (MAS) in L. edodes.

The molecular diversity analysis of Auricularia auricula-judae in China by nuclear ribosomal DNA intergenic spacer

Background: For the crossbreeding of Auricularia auricula-judae, selecting the appropriated parents in hybridization is very important. However, the classification and diversity analysis of A. auricula-judae has been equivocal, due to the similarity of the fruiting body morphology and its susceptibility to environmental influences. For this purpose, the molecular diversity of 32 A. auricula-judae commercial cultivars in China was analyzed by using the nuclear ribosomal DNA intergenic spacer. Results: The complete nuclear rDNA gene complex of A. auricula-judae isolate is 11,210 bp long, and contains the 18S, 5.8S, and 28S rRNA gene as well as the ITS and IGS regions. Based on the sequence data, four more effective primer combinations for the IGS region of A. auricula-judae were designed. Nucleotide sequence variation in the IGS among 32 A. auricula-judae commercial cultivars in China sorted into three strongly supported clades, which is correlated with geographical regions. Most strains originated from the same area were with a narrow genetic basis and could possibly be domesticated from the local wild-type strains. Conclusion: The grouping information obtained in the present work provides significant information for further genetic improvement in A. auricula-judae, and suggested that the IGS region can be used as an excellent tool for identification of genetic variation.