Table 1. Phenotypes of C. higginsianum pathogenicity mutants.

Mutant	VS 1	AP 2	PH 3	SH 4	Callose 5	ROS 6	MM 7
WT	+++	90%	39% a	3% a	11%	7%	100%
Group I: Mutants impaired in appressoria formation (<10% of conidia)
vir-1	0	0	0	0	0	-	++
vir-2*	0	0	0	0	0	0	0
vir-3	0	0	0	0	n.d.	n.d.	+
vir-4	0	0	0	0	0	-	+
vir-5	0	0	0	0	n.d.	n.d.	+
Group II: Mutants impaired in penetration or establishment of primary hyphae (AP formation >10%, PH formation ≤1%)
vir-10*	+	+++	0	0	-	-	++
vir-11*	0	++	0	0	n.d.	0	+
vir-12*	0	+++	0	0	-	0	++
vir-13	+	++	0	0	-	+	0
vir-14	0	++	0	0	-	0	++
vir-15*	0	+++	0	0	-	0	++
vir-16	0	+++	0	0	0	0	+
vir-17	0	+++	0	0	0	0	++
vir-18	0	+	0	0	0	0	++
vir-19	0	+++	0	0	-	0	++
vir-20	0	+++	0	0	-	0	++
vir-21	0	+++	0	0	-	0	++
vir-22	0	+++	0	0	-	-	++
vir-23	+	++	0	0	-	-	++
vir-24	0	+++	0	0	-	0	++
vir-25	+	+++	0	0	-	-	++
vir-26	0	+++	0	0	-	0	++
vir-27	0	+++	0	0	0	-	++
vir-28	0	+++	0	0	n.d.	0	++
Group III: Switch mutants (PH formation >10%, SH formation ≤1%)
vir-40*	0	++	++	0	-	-	++
vir-41*	+	+++	++	0	-	-	++
vir-42*	+	+++	++	0	0	++	++
vir-43*	0	+++	++	0	-	0	0
vir-44*	+	+++	++	0	-	0	++
vir-45*	+	+++	++	0	-	0	++
vir-46	+	+++	++	0	-	+	++
vir-47	+	+++	++	0	-	++	++
vir-48	+	+++	++	0	+	++	++
vir-49	+	+++	++	0	+	++	+
vir-50	+	+++	++	0	-	-	++
vir-51	+	+++	++	0	0	-	++
vir-52	+	+++	++	0	-	-	++
vir-53	0	+	++	0	0	++	++
vir-54	+	+++	+	0	-	0	++
vir-55	+	+++	++	0	0	-	++
vir-56	0	+	++	0	0	-	++
Group IV: Not classified
vir-70*	0	+++	+	+++	-	-	++
vir-71*	+	+++	+++	++	-	-	++
vir-72	+	++	+	0	-	++	++
vir-73*	0	+++	+	0	0	-	++
vir-74*	+	+++	++	+	-	+	++
vir-75	+	+++	+	0	-	++	++
vir-76	+	+++	+++	+	0	-	++
vir-77*	++	+++	+++	+++	-	++	++
vir-78	+	+++	+++	+	-	0	++
vir-79*	+	+++	+	+	-	-	++
vir-80	+	+++	++	+	-	+	++
vir-81	+	++	++	++	0	-	++
vir-82	++	+++	+++	+++	-	-	++
vir-83	+	+++	++	+++	-	++	++
vir-84	+	+++	++	+++	-	-	++
vir-85	+	+++	++	+	-	0	++
vir-86	+	+++	+	+	-	0	++
vir-87	++	+++	+++	+++	-	+	++
vir-88	+	+++	++	+	-	0	++
vir-89	+	+++	+	+	-	0	++
vir-90	+	+++	++	++	-	0	++
vir-91	+	+++	+	++	0	-	++
vir-92	++	+++	+++	+++	-	-	++
vir-93	++	+++	+++	+++	-	-	++
vir-94	++	+++	+++	+++	-	++	++
vir-95	+	+++	++	+	-	0	++
vir-96	+	+++	+	+	-	-	++
vir-97	+	+++	+	+	-	-	++
vir-98	0	+++	+	0	-	-	++
vir-99	+	+++	++	+	+	++	++
vir-100	+	+++	++	+	-	++	++
vir-101	0	+++	++	+	-	++	++
vir-102	++	+++	+++	WT	-	-	++
vir-103	0	++	+	0	-	-	++

* Mutant was generated by transformation with a plasmid other than pPK2. See Text S1.

^a Wild type data is given 3 days after infection since strong symptom development after 4 days prevented reliable quantification. After 4 days, the number of secondary hyphae ranged from >30 to >60%.

¹ Visible symptoms after 4 days of spray infection; 0: no visible symptoms. +: reduced symptoms, ++: slightly reduced symptoms, +++: WT.

² in vitro appressoria formation on 1,16-hexadecanediol-coated petri dishes in percentage of conidia; 0: <10%, +: 10–24%, ++: 25–50%, +++: >50%.

³ Formation of primary hyphae 4 days after spray-infection of A. thaliana Col-0 in percentage of appressoria; 0: <1%, +: 1–10%, ++: 11–25%, +++: >25%.

⁴ Formation of secondary hyphae 4 days after spray-infection of A. thaliana Col-0 in percentage of primary hyphae; 0: ≤ 1%, +: 1–10%, ++: 11–20%, +++: >20%.

⁵ Callose papilla (stained with aniline blue) formed beneath appressoria 3 days after spray-infection of A. thaliana Col-0 in percentage of appressoria; 0: <1%,-:1–10%, +: 11–12%, ++: >12%, n.d.: not detectable.

⁶ ROS accumulating cells (stained with 3,3-diaminobenzidine) beneath appressoria 3 days after spray-infection of A. thaliana Col-0 in percentage of total appressoria; 0: <1%,-: 1–5%, +: 6–7%, ++: >7%, n.d.: not detectable.

⁷ Colony diameter on Czapek-Dox minimal medium after 5 days compared to WT; 0: no growth, +: <30%, ++: >30%.