Properties analysis of transcription factor gene TasMYB36 from Trichoderma asperellum CBS433.97 and its heterogeneous transfomation to improve antifungal ability of Populus

Cloning of the TasMYB36 gene from T. asperellum CBS433.97 and bioinformatics analysis

The TasMYB36 gene was cloned from the T. asperellum CBS433.97 genome. Its DNA is 1146 bp in length and contains one intron. Its cDNA is 996 bp in length and encodes a protein comprising 331 aa. The isoelectric point (pI) of TasMYB36 protein is 10.25 and its molecular weight is 36.0 kDa. The protein was identified as an MYB transcription factor (Pfam 00249) using Pfam prediction, with one MYB DNA-binding domain at amino acids 15 to 57. The cDNA sequence was deposited in GenBank with the accession number KT834975.

Differential expression of TasMYB36 in response to five phytopathogen fermentation broths

The response of TasMYB36 was investigated by qRT-PCR after T. asperellum CBS433.97 was subjected to five biotic stresses; MM without phytopathogens was used as a control (Fig. 1a). TasMYB36 transcription was upregulated strongly at 4 h in response to the fermentation broths of A. alternate (Fig. 1b) and Cytospora chrysosperma (Fig. 1c), by 8.06 (2^3.01) and 7.78 (2^2.96)-fold, respectively. TasMYB36 transcription was also upregulated at 4 h in response to the fermentation broths of Fusarium oxysporum (Fig. 1d) and Rhizoctonia solani (Fig. 1e), by 1.18 (2^0.24) and 4.41 (2^2.14)-fold, respectively. However, TasMYB36 transcription was downregulated by the fermentation broths of Sclerotinia sclerotiorum (Fig. 1f), but was downregulated more strongly byMM at 4h, reaching to 8.46 (2^3.08)-fold. These results suggested that the TasMYB36 gene is closely associated with the response of T. asperellum CBS433.97 against biotic stresses.

Figure 1

The stress response of TasMYB36 of Trichoderma against five pathogen fermentation broths. (a) Mineral medium (MM) with 0.1% (w/v) glucose (as the control); (b–f) Five biotic stresses (5% (v/v) fermentation broths from A. alternate, C. chrysosperma, F. oxysporum, R. solani, and S. sclerotiorum).

The properties of 79 MYB transcription factors

The properties of 79 MYB transcription factors from six sequenced Trichoderma genomes were analyzed (Table 1). There were 14, 13, 12, 12, 15, and 13 MYB transcription factors being encoded by T. asperellum CBS433.97, T. harzianum CBS 226.95, T. virens Gv29-8, T. atroviride, T. reesei, and T. longibrachiatum ATCC18648, respectively. Fifteen MYB genes had no intron and the other MYB genes contained different numbers of introns (1–7). The pIs of MYB transcription factors showed large differences, ranging from 4.04 to 10.6, and there were totally 31 basic proteins (pI > 7) and 48 were acidic proteins (pI < 7). Their molecule weights ranged from 22.21 to 255.29 kDa. The 79 MYB transcription factors were classified into four major groups, including 1R-MYB, 2R-MYB, 3R-MYB, and 4R-MYB, based on the repeat numbers of their MYB DNA binding domains¹⁴. All 79 MYB transcription factors from six sequenced Trichoderma genomes contained 48 1R-MYB, 30 2R-MYB and only 1 3R-MYB (in T. harzianum), without 4R-MYB (Table 1). According the type and location of the MYB DNA binding domains, the MYB transcription factors could be divided in nine types, including Type B (n = 31), Type B₂ (n = 2), Type B₆ (n = 8), Type B₇ (n = 6), Type BB (n = 10), Type B₆B (n = 12), Type BB₆ (n = 6), Type B₆B₄ (n = 3), and Type B₆B₆B (n = 1). Most 1R-MYB transcription factors belonged to Type B and most 2R-MYB transcription factors belonged to Type BB and Type B₆B (Table 1).

Table 1

The properties of 79 MYB transcription factors from 6 sequenced Trichoderma genomes.

Gene Name	The Location of Gene	Introns	AA	pI	MW (kDa)	CD	Type
TasMYB25	scaffold_11:371466–372557 (−)	3	231	9.65	25.26	135–177/179–222
TasMYB27	scaffold_11:1343526–1344021 (−)	1	238	5.78	27.40	129–186/176–207
TasMYB36	scaffold_4:2116156–2117302 (−)	1	331	10.2	36.62	15–57
TasMYB38	scaffold_20:103014–103337 (−)	0	349	10.3	38.11	42–88/98–134
TasMYB58	scaffold_6:1378471–1380145 (+)	1	523	6.64	58.88	79–126
TasMYB62	scaffold_4:1210975–1213105 (−)	0	570	6.09	62.64	414–501
TasMYB67	scaffold_2:1420902–1422857 (−)	1	621	9.07	67.27	306–356/454–503
TasMYB75	scaffold_8:12464–15001 (−)	2	690	4.92	75.36	393–436
TasMYB86	scaffold_2:2509057–2511447 (−)	1	777	6.32	86.69	8–52/54–106
TasMYB92	scaffold_4:377301–380443 (−)	2	816	5.12	92.69	113–173
TasMYB118	scaffold_8:586973–590579 (−)	4	1089	5.33	118.05	591–647
TasMYB171	scaffold_3:1189064–1195065 (−)	6	1515	5.54	171.69	1079–1139/1147–1238
TasMYB182	scaffold_1:2715111–2722223 (+)	5	1657	8.99	182.38	671–715
TasMYB255	scaffold_4:2100911–2107305 (−)	1	2111	6.46	255.29	989–1032/1280–1323
ThaMYB22	scaffold_7:2283613–2284276 (−)	1	195	5.22	22.11	86–143/134–164
ThaMYB28	scaffold_18:68586–70065 (−)	5	263	9.21	28.85	162–206/208–253
ThaMYB34	scaffold_12:1162369–1163533 (−)	4	301	7.65	34.72	13–76/68–127/116–162
ThaMYB35	scaffold_4:2044751–2045862 (−)	1	326	10.4	35.88	13–57
ThaMYB36	scaffold_9:184232–184780 (+)	0	330	7.24	36.56	10–56/62–107
ThaMYB58	scaffold_10:864556–866677 (−)	1	523	6.47	58.95	79–126
ThaMYB62	scaffold_4:1415189–1416886 (+)	0	565	5.88	62.38	407–494
ThaMYB75	scaffold_30:20305–22916 (−)	2	691	4.90	75.22	392–437
ThaMYB68	scaffold_1:2082037–2083923 (−)	0	628	8.75	68.31	320–370/475–539
ThaMYB86	scaffold_1:3125036–3127439 (+)	1	777	5.72	86.83	9–69/58–102
ThaMYB113	scaffold_14:406732–410162 (−)	4	1049	5.37	113.88	582–638
ThaMYB173	scaffold_5:2004340–2010727 (+)	5	1542	5.32	173.62	1118–1178
ThaMYB239	scaffold_4:2029596–2037045 (−)	2	2145	6.91	239.70	1289–1334
TviMYB26	scaffold_20:475405–476732(−)	2	244	9.37	26.69	164–206
TviMYB27	scaffold_11:861800–862578 (−)	1	237	5.75	27.03	128–185/174–220
TviMYB36	scaffold_2:600988–602083 (+)	1	328	10.4	36.34	13–57
TviMYB37	scaffold_21:527963–529148 (−)	1	340	8.73	37.46	91–141
TviMYB42	scaffold_4:2202176–2203303 (−)	0	375	8.80	42.03	43–89/95–135
TviMYB57	scaffold_2:1224262–1225998 (−)	1	515	7.77	57.27	356–443
TviMYB61	scaffold_1:2235161–2236948 (−)	1	563	5.56	61.49	286–336/432–496
TviMYB70	scaffold_1:2459037–2460905 (−)	0	622	4.35	70.28	130–175
TviMYB73	scaffold_23:451456–453932 (+)	2	666	4.97	73.01	367–412
TviMYB86	scaffold_1:3347583–3349985 (+)	1	777	5.71	86.60	9–69/60–102
TviMYB110	scaffold_6:98720–101772 (−)	1	993	4.93	110.71	573–633
TviMYB243	scaffold_2:609834–616508 (+)	2	2175	7.76	243.66	1304–1349
TreMYB27	scaffold_9:1030437–1031225 (−)	1	235	5.87	27.03	126–183/172–218
TreMYB32	scaffold_1:1815585–1816698 (+)	2	292	10.3	32.37	13–57
TreMYB34	scaffold_5:317936–320047 (+)	4	318	8.94	34.45	164–208
TreMYB38	scaffold_10:782895–784125 (+)	2	349	8.99	38.32	96–166
TreMYB40	scaffold_28:181570–182670 (−)	0	336	9.62	40.30	43–89/95–135
TreMYB56	scaffold_1:767149–768965 (+)	1	514	9.08	56.63	355–442
TreMYB58	scaffold_24:241032–243046 (−)	1	514	6.29	58.95	79–126
TreMYB70	scaffold_2:333659–335521 (−)	0	620	4.43	70.36	133–178
TreMYB72	scaffold_2:61770–64535 (−)	0	669	6.57	72.02	363–413/510–574
TreMYB73	scaffold_2:1994423–1996847 (+)	3	661	5.07	72.19	361–406
TreMYB86	scaffold_2:1189091–1191556 (+)	1	777	6.01	86.34	9–69/58–102
TreMYB109	scaffold_26:242436–245500 (+)	1	988	5.07	109.39	566–626/614–718
TreMYB125	scaffold_11:581897–585735 (−)	5	1144	6.23	125.58	133–178
TreMYB186	scaffold_5:1293771–1300234 (−)	7	1074	9.03	186.65	738–783
TreMYB232	scaffold_1:1826274–1833439 (+)	5	2095	6.19	232.59	1018–1063
TatMYB27	scaffold_7:1531759–1533595 (+)	7	249	6.45	27.53	140–184
TatMYB28	scaffold_17:70061–70858 (+)	1	240	5.85	27.68	131–188/177–223
TatMYB33	scaffold_16:754009–754332 (−)	0	305	10.6	33.74	9–55/61–106
TatMYB36	scaffold_12:678408–679586 (−)	2	326	10.3	36.24	13–57
TatMYB37	scaffold_6:756022–757214 (−)	1	336	9.04	37.82	88–156
TatMYB56	scaffold_10:1254042–1256117 (−)	1	517	9.02	56.12	351–438
TatMYB65	scaffold_2:1998371–2000164 (+)	0	597	9.33	65.11	331–381/483–547
TatMYB72	scaffold_7:9103–11290 (−)	2	663	4.97	72.75	364–409
TatMYB86	scaffold_2:841485–843878 (−)	1	777	6.36	86.50	9–69/58–102
TatMYB163	scaffold_5:1479192–1484396 (−)	5	1497	6.78	163.54	913–984
TatMYB167	scaffold_3:1802095–1808280 (+)	7	1487	5.49	167.95	1084–1144
TatMYB241	scaffold_6:756022–757214 (−)	1	2176	5.81	241.48	1063–1108/1354–1399
TloMYB24	scaffold_8:1490083–1490650 (−)	1	263	5.66	24.11	103–160/149–196
TloMYB29	scaffold_1:2981637–2982199 (−)	4	265	9.06	29.15	165–209/211–256
TloMYB41	scaffold_3:2453950–2455526 (−)	0	375	9.76	41.20	43–89/95–135
TloMYB58	scaffold_6:1035137–1037465 (−)	1	520	6.29	58.49	79–126
TloMYB63	scaffold_3:1526425–1528647 (−)	0	582	6.16	63.42	423–510
TloMYB67	scaffold_2:2077891–2080313 (+)	2	609	6.04	67.27	9–69/58–102
TloMYB75	scaffold_2:2857988–2860562 (+)	2	690	4.04	75.04	391–436
TloMYB82	scaffold_2:1041805–1044054 (−)	0	749	6.56	82.31	368–418/520–584
TloMYB95	scaffold_6:296503–299477 (−)	2	848	5.34	95.92	111–171
TloMYB104	scaffold_14:670920–674674 (−)	3	1013	5.26	138.83	472–528
TloMYB109	scaffold_5:1468188–1471776 (−)	1	998	5.09	109.62	565–625/614–718
TloMYB138	scaffold_1:2050016–2056293 (+)	3	1259	8.97	138.85	288–333
TloMYB244	scaffold_3:536391–543984 (+)	2	2183	8.94	244.62	1310–1355

: MYB DNA-bind, : MYB DNA-bind 2, MYB DNA-bind 4, : MYB DNA-bind 6, : MYB DNA-bind 7. : Type1(B), : Type2(B2), : Type3(B₆), : Type4(B7) : Type5(BB), : Type6(B₆B), : Type7(BB₆), Type8(B₆B₄) : Type9(B₆B₆B), CD: conserved domain. AA: amino acid. MW: molecular weight.

The phylogenetics and alignment analysis of 79 MYB transcription factors

The phylogenetic analysis divided the 79 MYB transcription factors into 14 clades and the TasMYB36 protein studied in this paper was placed in Clade VII (Fig. 2). And the genetic distances of the 79 MYB transcription factors that occurs within each Trichoderma specie was calculated (Supplemental Table 2). For example, in the T. asperellum, TasMYB36 and TasMYB182 have the closest genetic relationship, the value was 1.223 (Supplemental Table 2), and TasMYB118 and TasMYB171 have the most remote genetic relationship, the value was 2.885 (Supplemental Table 2), the genetic relationship of different MYB transcription factors was consistent with their distribution in the phylogenetic tree (Fig. 2). The distribution of MYB proteins in the phylogenetic tree was closely related to their type and molecular weight. Most clades contained one MYB Type. Clades X and XIV contained two MYB Types, and clades XI and IV contained three MYB Types (Fig. 2). The MYB transcription factors only containing “MYB DNA-bind 2” or “MYB DNA-bind 7” were distributed in Clades XIII and VIII, respectively. The MYB transcription factors containing “MYB DNA-bind 4” were distributed in Clade IV. MYB proteins in the same clade shared the similar molecular weight, for example, six MYB proteins in the Clades XIV had the highest molecular weight (average 242.3kDa), and six MYB proteins in the Clades III had the lowest molecular weight (average 25.8kDa). MYB transcription factors in each clade were derived from different Trichoderma spp., but shared similar pI and number of introns (Table 1), Most MYB transcription factors in clade VI had no introns, but those in clades II, III, V, and XIII had one or two introns (Table 1). MYB transcription factors in clades II, III, IV, V, IX, X, XII, and XIII (Fig. 2) were acidic proteins (Table 1) and the pI of proteins in clade IX was the lowest (average 4.81 ± 0.77). MYB transcription factors in clades VI, VII, and XI (Fig. 2) were basic proteins (Table 1) and the pIs of proteins in clade VII were the highest, (average 9.82 ± 0.85).

Figure 2

Phylogenetic analysis of 79 MYB transcription factors. The 79 MYB transcription factors were obtained from six sequenced Trichoderma genomes, including T. asperellum CBS433.97, T. harzianum CBS 226.95, T. virens Gv29-8, T. atroviride, T. reesei, and T. longibrachiatum ATCC18648. They contained 14, 13, 12, 12, 15, and 13 MYB transcription factors, respectively. The phylogenetic tree was constructed using the neighbor-joining method in the MEGA6.0 program.

The 79 MYB transcription factors contained 113 MYB DNA binding domains, which were classified into five categories, including “MYB DNA-bind” (n = 72), “MYB DNA-bind 2” (n = 2), “MYB DNA-bind 4” (n = 3), “MYB DNA-bind 6” (n = 30), and “MYB DNA-bind 7” (n = 6). The proteins contained many more “MYB DNA-bind” and “MYB DNA-bind 6” domains than the other domains, and the alignment analysis showed the DNA-binding sites (highlighted in green or Bold) of MYB transcription factors in the same clade (Figs 3 and 4) were relatively conserved; however, those in different clades had obvious differences. According the distribution of DNA binding sites, the “MYB DNA-bind” domains were classified into two groups. The DNA binding sites of “MYB DNA-bind” in Group 1 contained 15 residues and there were three, three, and nine residues in α-helix1, α-helix2, and α-helix3, respectively. Blastp analysis at NCBI showed that the fourth amino acid of the DNA binding site was located in α-helix2 and was a basic amino acid; the seventh, tenth, twelfth, and thirteenth amino acids of the DNA binding site were located in α-helix3 and were also basic amino acid; and the eleventh amino acid was aspartic acid (D). The DNA binding sites of “MYB DNA-bind” in Group 2 contained 11 residues and there were 1 and 10 residues in α-helix1 and α-helix3, respectively (Fig. 3). The MYB transcription factors containing “MYB DNA-bind” in Group 1 were distributed in Clades I and XII of the phylogenetic tree (Fig. 2), which contained binding sites “RKW/G-WT/R-AGD-KDR-RT” and “RPW/P-WS/R-QVQ-KDK-RN”, respectively. The MYB transcription factors containing “MYB DNA-bind” in Group 2 were distributed in Clades II, III, IV, V, VI, VII, X, XI, and XIV (Fig. 2).

Figure 3

Alignment analysis of 72 “MYB DNA-bind”. The roman numerals indicate the location of the corresponding MYB proteins in the phylogenetic tree (Fig. 2). The DNA binding sites were predicted using BlastP at NCBI and are highlighted in green; however, the DNA binding sites of other “MYB DNA-bind” were not obtained; therefore, these DNA binding sites were predicted according to previous results and are highlighted in bold. “MYB DNA-bind” was divided in two groups and marked as G1 and G2.

Figure 4

Alignment analysis of 31 “MYB DNA-bind 6”. The roman numerals indicate the location of the corresponding MYB proteins in the phylogenetic tree (Fig. 2). The DNA binding sites were predicted using BlastP at NCBI and are highlighted in green; however, the DNA binding sites of other “MYB DNA-bind 6” were not obtained; therefore, these DNA binding sites were predicted according to previous results and are highlighted in Bold. “MYB DNA-bind 6” was divided in two groups and was marked as G1 and G2.

The “MYB DNA-bind 6” domains were also classified into two groups (Fig. 4). The DNA binding sites of “MYB DNA-bind 6” in Group 1 contained 12 residues and there were one, two, and nine residues in α-helix1, α-helix2, and α-helix3, respectively. Blastp analysis at NCBI showed that the second amino acid in front of conserved “W” residue in α-helix2 was a basic amino acid; the fourth, eighth, tenth, and eleventh amino acids of DNA binding sites were arginine (pI < 7) located in α-helix3. The DNA binding sites of “MYB DNA-bind 6” in Group 2 contained 11 residues and there were one and ten residues in α-helix1 and α-helix3, respectively (Fig. 4). The MYB transcription factors containing “MYB DNA-bind 6” in Group 1 were distributed in Clade I of the phylogenetic tree (Fig. 2), and contained the binding site “F-WS/T-R-PTD-RDR-RN”. The MYB transcription factors containing “MYB DNA-bind 6” in Group 2 were distributed in Clades II, III, IV, X, and XI (Fig. 2). The “D” in α-helix1 and “W” in α-helix2 were absolutely conserved. In addition, the C- and N-termini of the “MYB DNA-bind 6” domain contained the same conserved amino acid motif “W^T/_S-EEDE-L”, which was also present in the N-termini of the amino acid sequences of “MYB DNA-bind”.

Promoter analysis of 79 MYB genes from six sequenced Trichoderma genomes

The 1,000 bp upstream sequences of the 79 MYB genes from the six sequenced Trichoderma genomes were cloned. Using SCPD analysis, motifs were predicted and their locations were marked in the promoter regions of the MYB genes (Fig. 5). Most of the motifs were closely related to stress responses and detoxification, including GCR1, GCN4, ADR1, STRE, HSTF, PHO4, GC/FAR, PDR3, ABF1, CPF1, and so on. In particular, there were 240 GCR1s and 214 GCN4s motifs in the promoter regions of the 79 MYB genes. In addition, motifs GCR1, ADR1, HSTF and ABF1 are closely related to G-proteins and their receptors which are involved in sensing the oligopeptides secreted by phytopathogens³⁰, so these motifs play an important roles in upregulation of disease-resistant genes, synthesis of secondary metabolites and hyperparasitism. In addition, the type, density, and location of the motifs in MYB promoter regions were closely related to the position of the corresponding MYB transcription factors in the phylogenetic tree. The promoter sequences of 6 MYBs in Clade I contained many GCR1s and ADR1s motifs (average 4.8 and 6.2, respectively) (Fig. 5), which can improve the binding efficiency of transcription factors with both motifs, so these MYBs were closely related to signal transduction of Trichoderma in biocontrol process. The promoter sequences of 12 MYBs in Clade II and VIII contained more GCN4s (average 4.5) and STRE (average of 2.80) motifs than the other MYBs respectively, so they could mainly responded to biotic and abiotic stresses, and further upregulated defense response genes in Trichoderma. These STRE motifs were distributed mainly from −600 to −900 bp. All above indicated MYB transcription factors played important roles in Trichoderma adapting environment and survival.

Figure 5

Promoter analysis of 79 MYB genes. T. asperellum, T. harzianum, T. virens, T. reesei, T. atroviride, and T. longibrachiatum are abbreviated as Tas, Tha, Tvi, Tat, Tre and Tlo. The name of each MYB transcription factor is made up of three parts, including the abbreviation of the species name, MYB, and the molecular weight of the protein. The different colored markers indicate predicted different cis-motifs.

The construction of Populus transformants and TasMYB36 transcription detection

The plant transgenic vector pROKII-MYB36 was constructed (Fig. 6a) and transgenic Agrobacterium tumefaciens EHA105-MYB36 were obtained. The leaves of PdPap poplar were then infected with EHA105-MYB36 and 15 transformants were obtained by selecting with Kanamycin (50 mg/L); however, uninfected leaves did not generate resistant buds. PCR detection indicated that TasMYB36 from T. asperellum was integrated successfully in the Populus genome and three transformants (PdPap-TasMYB36s) were chosen for subsequent experiments. The qRT-PCR analysis showed that the TasMYB36 was transcribed in three PdPap-TasMYB36s, but not in PdPap-Con (Fig. 6b).

Figure 6

The construction of expression vector pROKII-TasMYB36, and transcription detection of TasMYB36 gene and plant hormone signal genes in PdPap-TasMYB36s. (a) The recombinant vector pROKII-MYB36. (b) Transcription detection of TasMYB36 gene in PdPap-TasMYB36s, 1–4: PdPap-Con, PdPap-TasMYB36-1, PdPap-TasMYB36-2, and PdPap-TasMYB36-3. (c,d) The transcription analysis of NPR1 and PR1 in the sialic acid (SA) pathway. (e,f) The transcription analysis of JAR1 and MYC2 in the jasmonic acid (JA) pathway.

Transcription analysis of PdPap-TasMYB36s hormone signal transduction genes related to biotic stress responses

The SA and JA pathways (KEGG: map04075) are two important signal transduction pathways related to stress responses. In the SA pathway, the PR1 as pathogenesis related gene can respond to biotic stresses and improve the antifungal ability of organisms, and the NPR1 protein could be the sole sensor from SA to PR gene expression in SA hormone signal transduction pathway³¹. In our study, the transcription of NPR1 and PR1 were upregulated in all three PdPap-TasMYB36s at 4 h under A. alternata stress, especially in PdPap-TasMYB36-1, in which the transcription of NPR1 and PR1 were upregulated by 2.60 (2^1.38)-fold and 15.89 (2^3.99)-fold respectively, and the transcription level of NPR1 and PR1 showed a positive correlation with each other (Fig. 6a,b). In the JA pathway, the JAR1 as jasmonic acid-amino synthetase could synthesize jasmonic acid (JA). JASMONATE ZIM-DOMAIN (JAZ) proteins could repress the expression of JA-response gene MYC2. However the perception of bioactive JA by the F-box protein COI1 (coronatine-insensitive protein 1) triggers the SCF^COI1/ubiquitin-dependent degradation of JAZ, and further enhance the expression of transcription factor gene MYC2. MYC2 can up-regulate others genes involved in a wide range of biological processes, including plant defense, secondary metabolism, and growth control^32,33. In our study, the JAR1 transcription in the PdPap-TasMYB36s was upregulated compared with PdPap-Con (Fig. 6c). Especially, JAR1 in PdPap-TasMYB36-2 was upregulated to the highest level, by 2.89 (2^1.53)-fold. The transcription of MYC2 in the PdPap-TasMYB36s was also upregulated compared with that in PdPap-Con. Especially, MYC2 in PdPap-TasMYB36-1 was upregulated to the highest level, by 2.95 (2^1.56)-fold (Fig. 6d).

Antifungal ability of the PdPap-TasMYB36s under A. alternate stress

Following the interaction of the PdPap-TasMYB36s with A. alternate, the antifungal ability of PdPap-TasMYB36s was studied by assessing the change in the POD, SOD, and CAT activities. SOD enzymes are responsible for decomposing the superoxide anion radical (O₂−) to H₂O₂ and O₂. peroxidase (POD) and catalase (CAT) further decompose H₂O₂³⁴. The POD activities of the three PdPap-TasMYB36s were higher than those of PdPap-Con and reached the highest level, approximately 37.0 U, at 5 × 10⁵ spores/mL. In addition, the POD activities of PdPap-Con decreased consistently, but the fluctuation of POD activities of PdPap-TasMYB36s was not obvious as the concentration of A. alternate spores increased (Fig. 7a).The POD activities of the three PdPap-TasMYB36s were higher than those of PdPap-Con and reached the highest level, approximately 37.0 U, at 5 × 10⁵ spores/mL. In addition, the POD activities of PdPap-Con decreased consistently, but the fluctuation of POD activity was not obvious as the concentration of A. alternate spores increased (Fig. 7a). The SOD activity of all PdPap-TasMYB36s was higher than that of PdPap-Con at all spore concentrations, and its activity reached the highest at 5 × 10⁵ spore/mL in all PdPap-TasMYB36s. At this spore concentration, the SOD activity of PdPap-Con was 51.7 U and that of PdPap-TasMYB36s were at least 61.1 U (Fig. 7b). The CAT activity of PdPap-Con ranged from 18.1 to 29.7 U, and the CAT activities of PdPap-TasMYB36s were higher at all spore concentrations, ranging from 20.2 to 36.3 U. The CAT activities increased with the increasing spore concentration (Fig. 7c).

Figure 7

The ability of transformants PdPap-TasMYB36s to resist infection by the pathogenic fungus A. alternate. (a–c) The POD, SOD, and CAT activities under A. alternate stress, respectively (student t-test p < 0.05). (d) The reactive oxygen content under A. alternate stress, 1–4: PdPap-Con, PdPap-TasMYB36-1, PdPap-TasMYB36-2, and PdPap-TasMYB36-3, respectively, A-C: three repeated experiments. (e) The ability of transformant PdPap-TasMYB36s to resist A. alternate infection. A1-C1 and A2-C2: leaves of the PdPap-Con. A3-C3, A4-C4, and A5-C5: the leaves of PdPap-TasMYB36-1, PdPap-TasMYB36-2, and PdPap-TasMYB36-3, respectively. The leaves A1-C1 were not inoculated with A. alternate, the others were inoculated with 5 × 10⁶ spores/mL A. alternate for 4 d. The inoculated positions on the leaves are shown on leaf A1. The three repeats of each poplar line were designated as A, B, and C. (f) The percentage of disease spot area, 1–4: PdPap-Con, PdPap-TasMYB36-1, PdPap-TasMYB36-2, and PdPap-TasMYB36-3.

The antifungal ability of PdPap-TasMYB36s was also studied using the ROS contents of poplar leaves under A. alternate stress (Fig. 7d). NBT staining revealed that the ROS content was lower in the PdPap-TasMYB36s leaves (smaller extent of blue staining) compared with that in the PdPap-Con leaves (larger extent of blue staining). PdPap-TasMYB36-2 had more blue staining compared with the other PdPap-TasMYB36s.

After PdPap-TasMYB36s and PdPap-Con were infected with A. alternate, the disease spots on leaves of PdPap-TasMYB36s were smaller (diameter: approximately 1–2 mm), showed regular circles, and the average of their area percentage was 0.63%, while the disease spots on the PdPap-Con leaves were larger (average diameter: 5–7 mm) and spread around, forming irregular shapes, even connected together (Fig. 7e), and the average of their area percentage was 24.7%. The leaf mortalities in the PdPap-TasMYB36-1, PdPap-TasMYB36-2, and PdPap-TasMYB36-3 were 33.3, 50, and 41.7%, respectively, whereas that in PdPap-Con reached 83.3%.

Strains, plasmids, and plant materials

T. asperellum CBS433.97 was obtained from the Agricultural Culture Collection of China and the phytopathogenic fungi Alternaria alternate was used for the pathogen-poplar interaction assay. Agrobacterium tumefaciens EHA105 and vector pROKII (Vaughan et al. 1987) were used for the genetic transformation of Populus davidiana × P. alba var. pyramidalis Louche (PdPap poplar). PdPap poplar seedlings were cultured aseptically in liquid rooting medium (MS medium with 1-Naphthaleneacetic acid (NAA) 0.25 mg/L and Sucrose 20 g/L) or differential medium (MS medium with NAA 0.05 mg/L, 6-Benzylaminopurine (6-BA) 0.5 mg/L, Sucrose 20 g/L and Agar 8 g/L) at 25 °C.

The cloning of the TasMYB36 gene and its responses to five kinds of fermentation broths stresses

The TasMYB36 was cloned using the sense primer (5′-ATGACACCTCACGTTCCAGA-3′) and the antisense primer (5′-TTATGCCAGTCTTCGCCTATG-3′). The transcription of TasMYB36 from T. asperellum CBS433.97 was detected by qRT-PCR under five biotic stresses: fermentation broths from A. alternate, C. chrysosperma, F. oxysporum, R. solani and S. sclerotiorum, respectively. The spores of T. asperellum were inoculated into 200 mL of 1/4 PD (Potato Dextrose) at a final concentration of 1 × 10⁴ spores/mL and cultured at 28 °C with continuous shaking at 200 rpm for 48 h. The mycelia were filtered, washed, and cultured in minimal medium (MM) for 2 h, and then transferred into the different MM inducing media (including the five fermentation broths mentioned above) for 48 h. For each inducing condition, mycelia were collected at 0, 4, 8, 12, 24, and 48 h post-induction. At each time point, 25 mL mycelium cultures per flask were collected, the biomass obtained in the three replicates and three 25 mL mycelium from three flasks under the same conditions were mixed and then stored at −80 °C. Total RNA was extracted from mycelia using the Trizol reagent (Invitrogen, USA), digested with DNaseI (Promega, USA), and reverse transcribed into cDNA using a PrimeScript RT Kit (Takara, Japan), according to the manufacturer’s instructions. The transcription level of TasMYB36 in T. asperellum was detected by qRT-PCR and calculated according to the 2^−ΔΔCt method^45,46, using cDNA as the template and Actin(T) as the reference gene. Three qRT-PCR replicates were performed for each cDNA sample. Primers for qRT-PCR (Supplemental Table 1) were designed using Primer 6.0 software (PREMIER Biosoft, USA).

Analysis of 79 MYB transcription factors from six sequenced Trichoderma genomes

Seventy-nine MYB genes were identified in the genomes of six sequenced Trichoderma, including T. asperellum CBS433.97, T. harzianum CBS226.95, T. virens Gv29-8, T. atroviride, T. reesei, and T. longibrachiatum ATCC18648 (http://genome.jgi.doe.gov/pages/tree-of-life.jsf). The conserved domains of the 79 MYB proteins were predicted using the BlastP program and analyzed using the Pfam program (http://pfam.sanger.ac.uk/). Furthermore, 1,000 bp sequences upstream of the transcription start site of these MYB genes were obtained from Trichoderma genomes and the motifs in the presumed promoters were predicted using the SCPD promoter analysis website (http://rulai.cshl.edu/SCPD/).

Phylogenetic and alignment analysis of MYB transcription factors from six sequenced Trichoderma genomes

A phylogenetic tree was constructed with amino acid sequences of 79 MYB transcription factors using the Neighbor-Joining method in MEGA6.0. And the genetic distances of 79 MYB was calculated with MEGA6.0. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated. There were 72 “MYB DNA-binding” and 30 “MYB DNA-binding 6” domains found in the 79 MYB proteins, and alignment analysis of both types of DNA-binding domains was carried out using Clustal Omega (http://www.ebi.ac.uk/services/proteins) and the sequence logo was drawn using Weblogo (http://weblogo.berkeley.edu/logo.cgi).

Construction of the plant expression vector pROKII-MYB36 and Populus transformation

To further detect the functions of TasMYB36, the gene was cloned by PCR using the sense primer (5′-ATCGTCTAGAATGACACCTCACGTTCCAGA-3′) containing an XbaI site and the antisense primer (5′-CGATGGTACCTTATGCCAGTCTTCGCCTATG-3′) containing a KpnI site. The PCR product of TasMYB36 and pROKII vector were double-digested with XbaI and KpnI, respectively, and ligated to generate vector pROKII-MYB36 (Fig. 6a). TasMYB36 was transformed into PdPap poplar using the Agrobacterium tumefaciens-mediated transformation system. Explants of PdPap poplar were cultured in differential medium containing 50 mg/L Kanamycin (used to select resistant buds), and 300 mg/L Cefotaxime sodium and 300 mg/L Ampicillin (used to eliminate Agrobacterium tumefaciens) at 25 °C for 15 d. Kanamycin-resistant buds were cut down and cultured in differential medium containing 30 mg/L Kanamycin for propagation. When the propagated seedlings grew to 3 cm in height, they were rooted in liquid rooting medium containing 30 mg/L Kanamycin, and finally transplanted into 10 cm pots containing a mixture of peat soil and vermiculite (5:1).

Transcription analysis of hormone signal transduction genes of PdPap-TasMYB36s

The transcription of hormone signal transduction genes (NPR1, PR1, JAR1, and MYC2) in the polar transformants (PdPap-TasMYB36s) under A. alternate stress were detected by qRT-PCR. A. alternate was inoculated in PD (Potato Dextrose) medium at 28 °C with continuous shaking at 200 rpm for 8 d and the fermentation broth was collected. The control PdPap-Con and three PdPap-TasMYB36s were induced in the fermentation broth for 24 h, respectively. Thereafter, 1 g of poplar leaves was collected from PdPap-Con and the three PdPap-TasMYB36s at 6, 12, and 24 h, and stored at −80 °C. Total RNA was extracted from poplar leaves using the cetyl trimethylammonium bromide (CTAB) method and reverse-transcribed into cDNA. The primers for qRT-PCR were the same as those used in our previous study⁴⁷. The qRT-PCR method was in accordance with description in the section of “The responses of the TasMYB36 gene under five biotic stresses”.

The detection of the ROS content and activity analysis of POD, SOD, and CAT

The PdPap-TasMYB36s and PdPap-Con were transplanted into soil and cultured for 40 d. Then, three concentrations of A. alternate spores suspensions were set, including 5 × 10⁶, 5 × 10⁵, and 5 × 10⁴ spores/mL, and each individual plant was treated with 50 mL of A. alternate spores suspension in the three replicates. After A. alternate spores and poplar were co-cultured for 5 d in soil, the top fifth leaves of PdPap-TasMYB36 and PdPap-Con were obtained and weighed using an analytical balance, before being ground in a mortar on the ice and centrifuged at 1100g for 10 min, respectively. Following the POD, SOD and CAT in supernatants were reacted for 30 min at 37 °C with the oxide respectively, the residual oxide in supernatants were reduced by another reductant and the supernatants showed a color by the interaction between reduzate and chromogenic agent. Finally, the content of reduzate was detected using spectrophotometer, and further worked out the activities of POD, SOD and CAT with the kit manufactured by nanjing jiancheng bioengineering institute (POD: A084-3, SOD: A001-1, and CAT: A007-1). The ROS levels in the leaves of PdPap-TasMYB36s were also analyzed. PdPap-TasMYB36s and PdPap-Con were cultured for 13 d in liquid rooting medium and the phytopathogen A. alternate was cultured in Potato Dextrose (PD) medium for 8 d. A. alternate mycelia were allowed to interact with the roots of the PdPap-Con and PdPap-TasMYB36s in sterile water for 48 h at 25 °C. Subsequently, the leaves of the PdPap-Con and PdPap-TasMYB36s were stained with 1 mg/mL nitroblue tetrazolium (NBT) overnight and destained in a solution containing 75% ethanol and 5% glycerol in a boiling water bath for 5 min.

The antifungal ability of PdPap-TasMYB36s against A. alternate

A. alternate was cultured in PDA medium at 28 °C, the spores were collected after 20 d and diluted to 5 × 106 spores/mL with sterile water. Leaves from PdPap-TasMYB36s and PdPap-Con were then infected by injection (two sites on either side of the main vein) with 5 μL of spore suspension, and cultured at 25 °C on wet filter paper in a petri dish. The relative area of disease spots were calculated by Chalkiness 1.0 program. Firstly, the infected leaves were scanned for digital images. Following the images inputted into Chalkiness 1.0 program, the location and profile of disease spots were recognized accurately, then the relative area of disease spots was evaluated⁴⁸.