Improved Powdery Mildew Resistance of Transgenic Nicotiana benthamiana Overexpressing the Cucurbita moschata CmSGT1 Gene

Abstract

Powdery mildew (PM), which is mainly caused by Podosphaera xanthii , is a serious biotrophic pathogen disease affecting field-grown and greenhouse-grown cucurbit crops worldwide. Because fungicides poorly control PM, the development and cultivation of PM-resistant varieties is critical. A homolog of SGT1 ( suppressor of the G2 allele of skp1 ), which encodes a key component of the plant disease-associated signal transduction pathway, was previously identified through a transcriptomic analysis of a PM-resistant pumpkin ( Cucurbita moschata ) inbred line infected with PM. In this study, we have characterized this SGT1 homolog in C. moschata , and investigated its effects on biotic stress resistance. Subcellular localization results revealed that CmSGT1 is present in the nucleus. Additionally, CmSGT1 expression levels in the PM-resistant material was strongly induced by PM, salicylic acid (SA) and hydrogen peroxide (H 2 O 2 ). In contrast, SA and H 2 O 2 downregulated CmSGT1 expression in the PM-susceptible material. The ethephon (Eth) and methyl jasmonate (MeJA) treatments upregulated CmSGT1 expression in both plant materials. The constitutive overexpression of CmSGT1 in Nicotiana benthamiana ( N. benthamiana ) minimized the PM symptoms on the leaves of PM-infected seedlings, accelerated the onset of cell necrosis, and enhanced the accumulation of H 2 O 2 . Furthermore, the expression levels of PR1a and PR5 , which are SA signaling transduction markers, were higher in the transgenic plants than in wild-type plants. Thus, the transgenic N. benthamiana plants were significantly more resistant to Erysiphe cichoracearum than the wild-type plants. This increased resistance was correlated with cell death, H 2 O 2 accumulation, and upregulated expression of SA-dependent defense genes. However, the chlorosis and yellowing of plant materials and the concentration of bacteria at infection sites were greater in the transgenic N. benthamiana plants than in the wild-type plants in response to infections by the pathogens responsible for bacterial wilt and scab. Therefore, CmSGT1 -overexpressing N. benthamiana plants were hypersensitive to these two diseases. The results of this study may represent valuable genetic information for the breeding of disease-resistant pumpkin varieties, and may also help to reveal the molecular mechanism underlying CmSGT1 functions.

Expression of Pumpkin CmbHLH87 Gene Improves Powdery Mildew Resistance in Tobacco

“…The subcellular localization of CmbHLH87 was assessed with a CmbHLH87-GFP fusion protein into Arabidopsis protoplasts under the control of the 35S CaMV promoter. The GFP signal in protoplasts producing GFP alone was detected in the cytoplasm and nucleus ( Figure 1) supported by Guo et al (2019), whereas the signal from the CmbHLH87-GFP fusion protein was detected exclusively in the nucleus.…”

“…Pumpkin seeds were germinated and the resulting seedlings were grown as previously described (Guo et al, 2018). Seedlings at the third-leaf stage were treated as previously described by Guo et al (2019). Seedlings were sprayed with a freshly prepared spore suspension (10 6 spores/ml), exogenous 1.5 mM H 2 O 2 , 100 mM SA, 100 mM ABA, 100 mM MeJA, 0.5 g/L Eth and 0.4mM NaCl.…”

“…Three biological triplicates were averaged and bars indicate standard error of the mean. **denotes significant differences between wild-type (WT) and transgenic plants at p < 0.01. localization of CmbHLH87 was determined based on the GFP signal, which was detected with the confocal fluorescence microscope (UltraVIEW VoX, Olympus, Japan) (Guo et al, 2019).…”

“…Conidia were collected from tobacco leaves naturally infected with Golovinomyces cichoracearum, which are main pathogen isolates of PM. The upper second leaf from the transgenic and WT seedlings at the fifth-leaf stage was sprayed with a spore suspension (10 6 spores/ ml) and contine to in vitro grow for 10 d (Guo et al, 2019). At 10 dpi, mildew development on each leaf disk was recorded, using the following scale: 0 = no visible mildew development, 1 = 0 to 5%, 2 = 6 to 25%, 3 = 26 to 50%, 4 = 51 to 75%, and 5 = > 76% of disk surface covered with mildew, as described by Ishii et al (2001).…”

“…The bacterial solutions were injected into the underside of leaves between the lateral veins with a syringe lacking a needle. After 6 d, the concentration of the bacteria at the injection sites was determined as previously described (Guo et al, 2019). The injection sites were sampled with a circular perforator (1cm diameter) and ground in aseptic water, then serially diluted to produce the 10 2 -fold, 10 3 -fold, and 10 4 -fold diluents.…”