Screening of pathogenicity-deficient Fusarium oxysporum mutants established by Agrobacterium tumefaciens-mediated transformation

Figures & data

Table 1. List of oligonucleotides used in this study

Primer	Sequence (5ʹ-3ʹ)	Application
RB-0a	GGCAATAAAGTTTCTTAAGATTGAATCCTGT	hiTAIL-PCR
RB-1a	ACGATGGACTCCAGTCCGGCCTGTTGCCGGTCTTGCGATGATTATCA
RB-2a	GTAATGCATGACGTTATTTATGAGATGGGTT
LAD 1-1	ACGATGGACTCCAGAGCGGCCGC(G/C/A)N(G/C/A)NNNGGAA
LAD 1-2	ACGATGGACTCCAGAGCGGCCGC(G/C/T)N(G/C/T)NNNGGTT
LAD 1-3	ACGATGGACTCCAGAGCGGCCGC(G/C/A)(G/C/A)N(G/C/A)NNNCCAA
LAD 1-4	ACGATGGACTCCAGAGCGGCCGC(G/C/T)(G/A/T)N(G/C/T)NNNCGGT
AC1	ACGATGGACTCCAGAG
Hyg-F	ATG AAA AAG CCT GAA CTC AC	hph sequence
Hyg-R	CTA TTC CTT TGC CCT CG
H-F1	CGTTATGTTTATCGGCACT	Probe for Southern blot
H-R1	TTGGCGACCTCGTATTGG
H520F	CAAGGTCGACAAAAGCACAAA	Gene FOIG_07731
H520R	CCTAATCCTCTTGCCCTTCC
H-2109F	CCAACGCTCTCTGAACCACT	Gene CEK26_004779
H-2109R	CAACATGGACAGCCTCTTGA
H-2040F	TCACAGAACGAGCGAGCTAA	Gene Fso1
H-2040R	TTCCACAAACGATCACCGTA
H-2026F	TGTGACCTATGGGCATCTGA	Gene FOIG_09359
H-2026R	CGTACGACAGGACCCAATCT
H-711F	GGAATGGGAATAAGCACCAA	Gene FOIG_00043
H711-R	TGTTTGATTTGTTGGCTGGA
FolCZF1–F	GAAAGTGCCATTGGGAGAGTA	Gene FolCZF1
FolCZF1–R	TTTTGTCCCGTTGGCAACT
Actin-F	GTTGGACTTGGGGTTGATGGG	Actin
Actin-R	CAAGCGTGGTATTCTCACTCTGC

Fig. 1 Effects of different concentrations of hygromycin B on the mycelial growth and conidial germination of Fusarium oxysporum.

Fig. 2 Single-factor comparison of Agrobacterium tumefaciens-mediated transformation of Fusarium oxysporum. The factors include the co-cultivation duration (a), the co-cultivation temperature (b), the OD₆₀₀ of A. tumefaciens (c), the conidium concentration of F. oxysporum (d), and the concentration of AS (e). The error bars represent the standard deviations of the repeats, and the different letters indicate significant differences between different treatments (P < 0.05)

Fig. 3 Green fluorescent of the WT strain, HS2 (a, b), and a transformed strain, HS2-2483 (c, d), of Fusarium oxysporum. A and C represent the bright field; B and D represent the fluorescent field

Fig. 4 Genetic stability of different transformants. Growth of WT strain HS2 and different transformants on PDA media without hygromycin (a). WT strain HS2 and related transformants after five generations without artificial selection on PDA media that contained 100 μg mL⁻¹ hygromycin (b)

Table 2. Sporulation number of partial transformants per unit area

Isolate Name	Log No. Conidia (No./cm^2)*	Isolate Name	Log No. Conidia (No./cm^2)*
HS2	3.92 ± 0.01 efg	HS2-534	3.32 ± 0.03 j
HS2-406	3.88 ± 0.01 efg	HS2-527	4.08 ± 0.04 cd
HS2-2109	2.06 ± 0.00 m	HS2-917	3.96 ± 0.05 e
HS2-100	4.16 ± 0.10 bc	HS2-2366	4.20 ± 0.01 b
HS2-1016	3.12 ± 0.03 k	HS2-2488	3.98 ± 0.01 de
HS2-2229	-	HS2-2340	4.18 ± 0.01 bc
HS2-520	3.48 ± 0.01 i	HS2-2483	4.17 ± 0.01 bc
HS2-2060	3.34 ± 0.05 j	HS2-4186	4.36 ± 0.01 a
HS2-2521	3.42 ± 0.05 ij	HS2-101	2.61 ± 0.04 l
HS2-311	3.83 ± 0.01 fg	HS2-2405	3.83 ± 0.06 g
HS2-511	4.20 ± 0.00 b	HS2-352	3.94 ± 0.02 ef
HS2-2525	3.66 ± 0.00 h	HS2-322	4.19 ± 0.03 b

* ‘-’ represents no spore produced; ‘±’ indicates ±SEs of three repeats; the different letters indicate a significant difference in sporulation number between different transformants at the P <0.05 level based on Duncan’s new multiple range test.

Fig. 5 Colony morphology of the WT strain, HS2 (a), and partial transformants (b–i). B to I represent transformant strains HS2-100, HS2-908, HS2-1017, HS2-511, HS2-527, HS2-532, HS2-711 and HS2-1015, respectively

Table 3. Pathogenicity test of partial mutants

Strains	Radicle Length (cm)^a	NT ^b	NI ^c
CK	3.50 ± 0.05 a	30	0
HS2	0.38 ± 0.06 l	30	20
HS2-2483	3.42 ± 0.05 a	30	15
HS2-2521	3.24 ± 0.14 a	30	8
HS2-2109	3.62 ± 0.16 a	30	0
HS2-406	3.11 ± 0.03 ab	30	9
HS2-100	2.64 ± 0.22 bcd	30	6
HS2-2060	2.55 ± 0.27 bcde	30	7
HS2-2340	2.50 ± 0.89 cdef	30	7
HS2-520	2.74 ± 0.23 defg	30	4
HS2-2366	2.17 ± 0.14 defg	30	6
HS2-1016	1.91 ± 0.29 efgh	30	3
HS2-311	1.92 ± 0.24 efgh	30	6
HS2-2525	1.86 ± 0.14 fgh	30	4
HS2-511	1.75 ± 0.31 ghi	30	7
HS2-4186	1.38 ± 0.28 hij	30	12
HS2-527	0.64 ± 0.02 kl	30	18
HS2-352	0.62 ± 0.05 kl	30	14
HS2-2052	0.29 ± 0.07 lm	30	19
HS2-2084	0.22 ± 0.04 m	30	22

^a‘±’ indicates ±SEs of three repeats; the different letters indicate significant differences in radicle length between different transformants at the P <0.05 level based on Duncan’s new multiple range test.

^bN_T indicates the total number of germinated crabapple seeds.

^cN_I indicates the number of infected germinated crabapple seeds whose radicle colour turned brown.

Fig. 6 Comparison of the length of crab apple radicles between the WT strain HS2 (a) and the pathogenicity-deficient transformants (b, c, d). B, C and D represent transformant strains HS2-2483, HS2-2521 and HS2-2109, respectively

Fig. 7 Southern blot analyses of 8 different transformants of Fusarium oxysporum HS2 generated by ATMT. M represents the DNA marker; P represents the positive control of pCamhybgfp; Lanes 1–8 represent the transformant strains HS2-2483, HS2-2521, HS2-520, HS2-1106, HS2-2109, HS2-511, HS2-311 and HS2-2460, respectively; Lane WT represents the WT strain of F. oxysporum, HS2

Table 4. Analysis of identified genes possibly involved in the deficient pathogenicity of Fusarium oxysporum mutants

Mutant	Site of insertion	Tagged gene^a	Putative protein	Conserved protein	Signal peptide
HS2-711	Exon	FOIG_00043	No	No	No
HS2-2109	Upstream	CEK26_004779	No	No	No
HS2-2026	Exon	FOIG_09359	Benzoate 4-monooxygenase	No	No
HS2-520	Exon	FOIG_07731	No	No	No
HS2-2040	Upstream	Fso1	www domain protein	ABQ88348	No

^aDerived by BLAST searches of the NCBI database.

Fig. 8 RT-PCR analysis of the transcription of FOIG_07731, FOIG_00043, FOIG_09359, Fso1 and CEK26_00479. The β-actin gene was amplified and used as a control. Lane M represents the DNA marker; Lanes 2–6 represent different transformants mediated by ATMT; Lane 1 represents the WT strain of Fusarium oxysporum, HS2; Lane N represents the negative control

Fig. 9 RT-PCR analysis of the transcription of folczf1 in the different mutants. The β-actin gene was amplified and used as a control; Lanes 2 to 6 represent different transformants mediated by ATMT; Lane 1 represents the WT strain of Fusarium oxysporum, HS2; Lane N represents the negative control