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.

本研究对棉花（Gossypium barbadense，亦称皮马棉）根部的转录组响应进行了RNA测序分析，以探究由Fusarium oxysporum f.sp. vasinfectum（FOV）4号生理小种（FOV4）引起的感染。实验所用的棉花材料包括对FOV4易感的自交系Pima S-7（PI 560140）和Pima 3-79，以及具有FOV4抗性的自交系Pima S-6（PI 608346）。制备cDNA文库并进一步进行RNA测序分析所用的FOV4分离株（FOV CA-14）来自加利福尼亚州圣华金谷地弗雷斯诺县的自然感染地块。实验设计包括易感自交系Pima S-7（PS7）和Pima 3-79（P379）对FOV4的易感性，以及Pima-S6（PS6）对FOV4的抗性，分别用于cDNA文库的制备及RNA测序分析。控制组未进行接种处理，FOV4接种组和未接种对照组均进行三次重复实验。单个分生孢子培养物在-20°C的滤纸上储存。制备接种剂时，将分离株在含有每升3mM链霉素的20ml马铃薯葡萄糖琼脂（PDA）培养基中，于室温下培养一周。然后，从新鲜培养物中切取2-3mm²的小块，放入含有3mM链霉素的500ml马铃薯葡萄糖液体培养基中，在28°C下旋转培养3-4天。随后，通过八层纱布过滤分生孢子悬浮液以去除菌丝，并使用血细胞计数器进行定量，用水稀释至每毫升含1 x 10^6个分生孢子。将品种或基因型在发芽盘中培养一周，然后拔起幼苗。用自来水冲洗根部，立即将幼苗浸入含每毫升1 x 10^6个分生孢子的FOV4接种剂水悬液中3分钟。未接种对照组的处理方式相同，只是将幼苗浸入不含FOV4的水中。然后将幼苗移植到新盆中，每个盆中种植五株，置于维持24-28°C的暖性温室中。接种后11天（dai），将植株和土壤球从盆中取出，并在持续水流下轻轻洗去根部周围的土壤。清洗后的根部立即置于50ml螺口塑料试管中，加入液氮，并将试管置于-80°C的冰箱中，开口朝上，以便液氮蒸发。