Dissecting functions of KATANIN and WRINKLED1 in cotton fiber development by virus-induced gene silencing

Abstract

Most of the world's natural fiber comes from cotton (Gossypium spp.), which is an important crop worldwide. Characterizing genes that regulate cotton yield and fiber quality is expected to benefit the sustainable production of natural fiber. Although a huge number of expressed sequence tag sequences are now available in the public database, large-scale gene function analysis has been hampered by the low-efficiency process of generating transgenic cotton plants. Tobacco rattle virus (TRV) has recently been reported to trigger virus-induced gene silencing (VIGS) in cotton leaves. Here, we extended the utility of this method by showing that TRV-VIGS can operate in reproductive organs as well. We used this method to investigate the function of KATANIN and WRINKLED1 in cotton plant development. Cotton plants with suppressed KATANIN expression produced shorter fibers and elevated weight ratio of seed oil to endosperm. By contrast, silencing of WRINKLED1 expression resulted in increased fiber length but reduced oil seed content, suggesting the possibility to increase fiber length by repartitioning carbon flow. Our results provide evidence that the TRV-VIGS system can be used for rapid functional analysis of genes involved in cotton fiber development.

Figure 1.

sTRV-induced silencing of anthocyanidin and PA biosynthetic genes ANS and ANR. A to F, Plants infiltrated with empty vector control (CK), sTRV:ANS, and sTRV:ANR showed different phenotypes in systemic leaf (A), stem (B), root (C), flower bud (D), sliced flower bud (E), and fiber (F). V, Visual image (top); S, DMACA staining (bottom); BS, before staining but after decoloration (middle of E). Bars = 10 mm except in F, where bar = 2 mm. G, Relative transcript levels of ANS and ANR in systemic leaves of plants infiltrated with sTRV:ANS and sTRV:ANR. Control value was set as 1. Error bars represent sd (n = 3 biological replicates).

Figure 2.

psTRV-induced silencing of KTN. A and B, Plants infiltrated with empty vector control (CK) and psTRV:KTN showed different phenotypes in flower bud (A) and flower (B). Bars = 10 mm. C, Scanning electron micrograph of CK and psTRV:KTN sepals (top) and analysis of trichome branch numbers and trichome length in CK and psTRV:KTN sepals (bottom). Bars = 200 µm. D, Scanning electron micrograph of CK and psTRV:KTN ovule at 4 dpa. Bars = 100 µm. E, CK and psTRV:KTN ovules from cotton bolls (inset at right bottom) at 8 dpa. Bars = 10 mm. F, Relative KTN transcript levels in systemic leaves and ovules of plants infiltrated with CK and psTRV:KTN. CK value was set as 1. Error bars represent sd (n = 3 biological replicates). G, Immunolabeling of microtubule cytoskeleton of CK and psTRV:KTN fiber at 12 dpa. Arrows and circles delineate the disorganization of cortical microtubules. Bars = 10 µm.

Figure 3.

Silencing of KTN caused shorter cotton fibers and increased seed oil content. A, Phenotypes of empty vector control (CK) and KTN-silenced cotton bolls. Bar = 10 mm. B, Phenotypes of CK and KTN-silenced cotton fibers. Bar = 10 mm. C, Analysis of fiber length of CK and KTN-silenced cotton bolls (P < 0.001). D, Analysis of seed oil content (the ratio of raw oil weight to dry endosperm weight) in CK and KTN-silenced cotton seed (P < 0.05). [See online article for color version of this figure.]

Figure 4.

Silencing of WRI1 is a positive regulator of oil biosynthesis. A, Phenotypes of empty vector control (CK) and WRI1-silenced cotton bolls and seeds. Bars = 10 mm. B, Analysis of fiber length of CK and WRI1-silenced cotton bolls (P < 0.001). C, Relative gene transcript levels in CK and WRI1-silenced cotton seeds. Error bars represent sd (n = 3 biological replicates). D, Seed weight (left) and oil content (the ratio of raw oil weight to dry endosperm weight; right) of CK and WRI1-silenced cotton seeds. E, Fatty acid composition of CK and WRI1-silenced cotton seeds. [See online article for color version of this figure.]