Cotton (Gossypium barbadense) root transcriptional response to Fusarium oxysporum f.sp. vasinfectum (FOV) race 4 (FOV4) infection

We performed RNA-seq analysis of the root transcriptional response to Fusarium oxysporum f.sp. vasinfectum (FOV) race 4 (FOV4) infection in Gossypium barbadense, also known as Pima cotton. Susceptible Gossypium barbadense inbred lines Pima S-7 (PI 560140) and Pima 3-79 susceptible to Fusarium wilt [Fusarium oxysporum f.sp. vasinfectum (FOV)] race 4 (FOV4), and Pima S-6 (PI 608346) which is resistant to FOV4 infection, were used for the preparation of cDNA libraries and further RNA-seq analyses. An isolate of FOV4 (FOV CA-14) from a naturally infested field in Fresno County in the San Joaquin Valley, California was used in this study. Overall design: Susceptible Gossypium barbadense inbred lines Pima S-7 (PS7) and Pima 3-79 (P379) susceptible to FOV4 and Pima-S6 (PS6), resistant to FOV4, were used for the preparation of cDNA libraries/RNAseq analyses. An isolate of FOV4 (FOV CA-14) from a naturally infested field in Fresno County in the San Joaquin Valley, California was used in this study. We used no inoculation for control plants, three replicates for FOV4-inoculated and non-inoculated control, respectively. Cultures from single spores were stored on filter paper at -20oC. To produce inoculum, the isolate was grown in 9-cm-diameter Petri dishes containing 20 ml potato dextrose agar (PDA) with 3 mM streptomycin per liter at room temperature for 1 week. Then a few patches of 2-3-mm-squre of fresh grown culture were cut and put into 500 ml flasks containing potato dextrose liquid medium with 3 mM streptomycin at 28 C with rotation for 3-4 days. The conidial suspension was then filtered through eight layers of cheesecloth to remove hyphae, quantified with the aid of a hemacytometer, and diluted with water to obtain 1 x 10^6 conidia per ml. Cultivars or genotypes were germinated in seedling trays for one week and then seedlings were uprooted. Roots were rinsed with tap water and immediately dipped for 3 min into an aqueous suspension of FOV4 inoculum containing 1 x 10^6 conidia per ml. The non-inoculated control plants were processed the same way except they were dipped into water without FOV4. Seedlings were then transplanted into steam-sterilized U.C. Mix #2 (Baker, 1957) soil in new pots with five plants in each pot and placed in a warm greenhouse maintained between 24-28 degrees Celcius. At 11 days after inoculation (dai), the plant and soil ball were removed from the pots, and the soil removed gently from roots by washing under continuous water flow. The washed roots were placed immediately in 50ml screw-top plastic vials, liquid nitrogen was added, and the vials placed in a -80o C freezer with the tops off to allow vaporization of the liquid N.