Overexpression of Salicylic Acid Carboxyl Methyltransferase (CsSAMT1) Enhances Tolerance to Huanglongbing Disease in Wanjincheng Orange (Citrus sinensis (L.) Osbeck)

Abstract

Citrus Huanglongbing (HLB) disease or citrus greening is caused by Candidatus Liberibacter asiaticus (Las) and is the most devastating disease in the global citrus industry. Salicylic acid (SA) plays a central role in regulating plant defenses against pathogenic attack. SA methyltransferase (SAMT) modulates SA homeostasis by converting SA to methyl salicylate (MeSA). Here, we report on the functions of the citrus SAMT (CsSAMT1) gene from HLB-susceptible Wanjincheng orange (Citrus sinensis (L.) Osbeck) in plant defenses against Las infection. The CsSAMT1 cDNA was expressed in yeast. Using in vitro enzyme assays, yeast expressing CsSAMT1 was confirmed to specifically catalyze the formation of MeSA using SA as a substrate. Transgenic Wanjincheng orange plants overexpressing CsSAMT1 had significantly increased levels of SA and MeSA compared to wild-type controls. HLB resistance was evaluated for two years and showed that transgenic plants displayed significantly alleviated symptoms including a lack of chlorosis, low bacterial counts, reduced hyperplasia of the phloem cells, and lower levels of starch and callose compared to wild-type plants. These data confirmed that CsSAMT1 overexpression confers an enhanced tolerance to Las in citrus fruits. RNA-seq analysis revealed that CsSAMT1 overexpression significantly upregulated the citrus defense response by enhancing the transcription of disease resistance genes. This study provides insight for improving host resistance to HLB by manipulation of SA signaling in citrus fruits.

Overexpression of a “Candidatus Liberibacter Asiaticus” Effector Gene CaLasSDE115 Contributes to Early Colonization in Citrus sinensis

“…Plant SAR resistance plays important roles in citrus tolerance to HLB ( Zou et al, 2021 ), with SA, MeSA, and JA being essential signals for plant SAR responses ( Park et al, 2007 ; Zhu et al, 2014 ). To understand the effects of mSDE115 overexpression on SAR response, we first determined changes in the levels of SA, MeSA, and JA for OE1, OE6, and OE8 transgenic plants which were more susceptible to Ca Las ( Figure 7A ).…”

“…Compared with the WT control, SA and JA levels in all the transgenic lines were downregulated after overexpressing mSDE115 while MeSA content was not affected by the overexpression. Furthermore, the expression of nine SAR-associated genes, including three CsNPR3 genes ( CsNPR3-73 for Ciclev10017873m, CsNPR3-15 for Ciclev10031115m, CsNPR3-49 for Ciclev10031749m) and one CsNPR4-08 (Ciclev10033908m) ( Wang et al, 2016 ), and CsPR1 , CsPR2 , CsPR5, CsWRKY45 , and CsWRKY70 ( Zou et al, 2021 ) in transgenic plants were investigated by qRT-PCR. The results showed that the expression levels of all the PR and WRKY genes were significantly lower in OE1, OE6 and OE8 transgenic lines compared with the control.…”

“…The SA, methyl salicylate (MeSA) and jasmonic acid (JA) content in the supernatant was determined using plant enzyme-linked immunosorbent assay (ELISA) kits (Jiweibio, Shanghai, China) ( Zou et al, 2021 ). Briefly, the supernatant was first diluted with a 5 × ELISA diluent buffer before adding 50 μL of the diluted supernatant to reaction wells, followed by 100 μl of the detection antibody (horseradish peroxidase-conjugated antibody).…”

Citrus Genetic Transformation: An Overview of the Current Strategies and Insights on the New Emerging Technologies

“…Recently, several citrus genes that control traits of interest have been cloned and characterized as novel targets for cis and intragenesis approaches. Between them CsSAMT1 ( Salicylic acid Carboxyl Methyltransferase 1 ), which confers tolerance to HLB; CsMADS5, a fruit ripening-associated transcription factor, that positively regulates carotenoid biosynthesis in citrus; a CsMYB96 transcription factor, which enhances citrus fruit resistance against fungal pathogens; CiMADS43, a MADS-Box gene involved in citrus flowering and leaf development and CiNPR4, an NPR1-like gene that enhanced resistance of transgenic citrus plants to HLB ( Li et al, 2020 ; Khan et al, 2021 ; Lu et al, 2021 ; Peng et al, 2021b ; Zeng et al, 2021 ; Zhang et al, 2021 ; Zou et al, 2021 ).…”

Overexpression of CsSAMT in Citrus sinensis Induces Defense Response and Increases Resistance to Xanthomonas citri subsp. citri

“…In addition, CrSAMT -transgenic plants increased MeSA volatilization, promoting the induction of PR1 in distal tissues and in neighboring wild-type plants ( Gómez et al, 2020 ). Recently, overexpression of CsSAMT1 (homologous to CrSAMT ) in Wanjincheng oranges ( C. sinensis ) was demonstrated to enhance the tolerance to Huanglongbing caused by Candidatus Liberibacter asiaticus (LAS) in citrus species ( Zou et al, 2021 ).…”