Identification and functional analysis of cassava DELLA proteins in plant disease resistance against cassava bacterial blight

Accepted Manuscript Identification and functional analysis of cassava DELLA proteins in plant disease resistance against cassava bacterial blight Xiaolin Li, Wen Liu, Bing Li, Guoyin Liu, Yunxie Wei, Chaozu He, Haitao Shi PII:

S0981-9428(17)30423-0

DOI:

10.1016/j.plaphy.2017.12.022

Reference:

PLAPHY 5086

To appear in:

Plant Physiology and Biochemistry

Received Date: 20 September 2017 Revised Date:

8 December 2017

Accepted Date: 12 December 2017

Please cite this article as: X. Li, W. Liu, B. Li, G. Liu, Y. Wei, C. He, H. Shi, Identification and functional analysis of cassava DELLA proteins in plant disease resistance against cassava bacterial blight, Plant Physiology et Biochemistry (2018), doi: 10.1016/j.plaphy.2017.12.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Identification and functional analysis of cassava DELLA proteins in plant

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disease resistance against cassava bacterial blight

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Xiaolin Li1,#, Wen Liu2,#, Bing Li1, Guoyin Liu1, Yunxie Wei1, Chaozu He1,*, Haitao Shi1,*

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Biology, Institute of Tropical Agriculture and Forestry, Hainan University, Haikou, 570228,

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China

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Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresources and College of

Key Laboratory of Three Gorges Regional Plant Genetics & Germplasm Enhancement

(CTGU)/ Biotechnology Research Center, China Three Gorges University, Yichang, Hubei,

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443002, China

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#

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*

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These authors contributed equally to this work.

Corresponding author ([email protected] or [email protected])

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Running title: MeDELLAs regulate disease resistance

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Manuscript information: With 7 Figures and 3 Supplemental Tables

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List of author’s last names: Xiaolin Li, Wen Liu, Bing Li, Guoyin Liu, Yunxie Wei,

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Chaozu He, Haitao Shi

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Authors contributions: H Shi conceived and directed this study, wrote and revised the

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manuscript; Y Wei, W Liu B Li, G Liu and Y Wei performed the experiments, analyzed the

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data and revised the manuscript; C He provided suggestions and revised the manuscript.

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ACCEPTED MANUSCRIPT Abstract

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Gibberellin (GA) is an essential plant hormone in plant growth and development as well as

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various stress responses. DELLA proteins are important repressors of GA signal pathway. GA

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and DELLA have been extensively investigated in several model plants. However, the in vivo

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roles of GA and DELLA in cassava, one of the most important crops and energy crops in the

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tropical area, are unknown. In this study, systematic genome-wide analysis identified 4

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MeDELLAs in cassava, as evidenced by the evolutionary tree, gene structures and motifs

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analyses. Gene expression analysis found that 4 MeDELLAs were commonly regulated by

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flg22 and Xanthomonas axonopodis pv manihotis (Xam). Through overexpression in

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Nicotiana benthamiana, we found that 4 MeDELLAs conferred improved disease resistance

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against cassava bacterial blight. Through virus-induced gene silencing (VIGS) in cassava, we

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found that MeDELLA-silenced plants exhibited decreased disease resistance, with less callose

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deposition and lower transcript levels of defense-related genes. This is the first study

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identifying MeDELLAs as positive regulators of disease resistance against cassava bacterial

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blight.

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Key words: cassava (Manihot esculenta), cassava bacterial blight, DELLA, disease

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resistance, VIGS

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ACCEPTED MANUSCRIPT 1. Introduction

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Cassava (Manihot esculenta) is one of the major food crops in tropical areas (Wang et al.,

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2014; Wei et al., 2017). However, cassava is highly sensitive in bacterial blight such as

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Xanthomonas axonopodis pv. manihotis (Xam) (Lopez et al., 2005; Munoz-Bodnar et al.,

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2014), which is the causal agent of cassava bacterial blight. The typical symptoms of cassava

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bacterial blight include leaf wilting and angular leaf spots, leading to reduced food production.

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Thus, identification of cassava disease-resistant genes and the use of disease-resistant

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varieties by modern biotechnology breeding are of great importance to improve cassava

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production and develop the health and sustainable cassava industry.

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At least 136 gibberellins (GAs) have been identified in plants, bacteria and fungi (Davière

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and Achard, 2016; Hedden and Thomas, 2012). Most of the GAs are de-activated metabolites

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or precursors, while only few of them such as GA1, GA3, GA4 and GA7 are bioactive with

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biological activity (Hedden and Thomas, 2012). DELLA proteins are important repressors of

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GA signal pathway (Fukazawa et al., 2014). The roles of AtDELLAs have been widely

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revealed. On one hand, DELLA protein levels are directly maintained by GA receptor via the

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ubiquitin system for protein degradation. On the other hand, DELLAs interact with multiple

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transcription factors (ABA INSENSITIVE 3 (ABI3) and ABI5 in abscisic acid (ABA)

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signaling, BRASSINAZOLE-RESISTANT 1 (BZR1) in brassinosteroid (BR) signaling,

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PHYTOCHROME INTERACTING FACTORs (PIFs) in light signaling, AUXIN

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RESPONSE FACTOR 6 (ARF6) in auxin signaling, ETHYLENE-INSENSITIVE 3 (EIN3)

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in

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WD/bHLH/MYB complex and MYC2 in jasmonic acid (JA) signaling, B-ARRs in cytokinin

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(CK) signaling, DWARF 14 (D14) in strigolactone (SL) signaling, FLOWERING LOCUS C

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(FLC), CONSTANS (CO), etc) to participate in complex crosstalks of plant hormones

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(Davière and Achard, 2016; Gao et al., 2011; Hou et al., 2010; Li et al., 2015, 2016;

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Lozano-Juste and León, 2011).

(ET)

signaling,

JASMONATE-ZIM-DOMAIN

PROTEINs

(JAZs),

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GA is first identified as a key regulator of plant growth and development, such as the

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promotion of seed germination, leaf expansion, stem elongation, flowering and fruit

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development (Davière and Achard, 2016). Further studies suggested that GA is also widely

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involved in plant stress response (Davière and Achard, 2016; Gao et al., 2011; Qin et al., 3

ACCEPTED MANUSCRIPT 2014). AtDELLA proteins are reported to be involved in plant response to bacteria and fungi

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(Tan et al., 2014). For instance, Atdella mutants exhibit up-regulated expression of salicylic

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acid (SA)-related genes and down-regulated expression of JA-responsive genes, leading to

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enhanced resistance to pathogenic bacteria (Pst DC3000) and decreased resistance to fungus

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Alternaria alternata (Navarro et al., 2008). In addition, DELLA proteins also confer

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improved plant resistance to abiotic stresses, such as low phosphate, salt stress and cold stress

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(Achard et al., 2006, 2008b; Yang et al., 2013a, b). Mutation in OsGID1 shows increased

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resistance to rice blast infection, with elevated GA contents (Yang et al., 2013a, b). OsEUI1,

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encoding a cytochrome P450 oxygenase, is involved in basic resistance to rice bacterial blight

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and blast as well as salt tolerance through degrading bioactive GA molecules (Yang et al.,

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2013a, b). Recently, De Vleesschauwer et al. (2016) found that OsSLR1 is a positive

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regulator of hemibiotroph resistance by integrating SA- and JA-dependent defense signaling.

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To date, DELLA proteins have been widely identified in plants, including DWARF8 (D8)

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and DWARF9 (D9) in maize (Lawit et al. 2010), REDUCED HEIGHT-1 (RHT-1) in wheat

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(Peng et al., 1999), SLENDER RICE1 (SLR1) in rice (Ikeda et al., 2001), PROCERA in

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tomato (Martí et al., 2007), VvGAI1 in grapevine (Zhong and Yang, 2012). In Arabidopsis,

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there are five DELLAs, GA-INSENSITIVE (GAI), REPRESSOR OF ga1-3 (RGA),

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RGA-LIKE1 (RGL1), RGL2 and RGL3 (Achard et al., 2006, 2008a, b). However, no

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DELLA has been identified in cassava, one of the most important crops and energy crops in

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the tropical areas (Camilo et al., 2005; Muñoz-Bodnar et al., 2014).

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To extend our understanding of the in vivo roles of DELLA in cassava, a systematic

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identification of MeDELLAs were performed in this study. Moreover, 4 MeDELLAs were

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cloned and functionally analyzed, especially their possible roles in defense response against

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cassava bacterial blight.

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2. Materials and Methods

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2.1. Plant materials and growth conditions

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The cassava of South China 124 (SC124) variety was used in this study. The segment cuts

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from SC124 stems were cultivated in soil in the green house for about 30 days before sample

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harvest. The green house was controlled at 12 h light/28°C and 12 h dark/26°C cycles, with 4

ACCEPTED MANUSCRIPT the irradiance of 120-150 µmol quanta m-2 s-1. Hoagland’s solution was watered twice every

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week.

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2.2. Identification and comprehensive analyses of MeDELLAs

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The gene and protein sequences of MeDELLAs were downloaded from the Phytozome v10.3

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(http://www.phytozome.net/cassava.php), and further examined and confirmed by National

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Center for Biotechnology Information (NCBI)’s conserved domain database (CDD)

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(http://www.ncbi.nlm.nih.gov/cdd) (Marchler-Bauer et al., 2015) and Pfam database

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(http://pfam.xfam.org) (Finn et al., 2016). The coding sequences (CDS) of AtDELLAs and

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OsSLR1 were obtained from The Arabidopsis Information Resource (TAIR) v10

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(http://www.Arabidopsis.org)

Rice

Genome

Annotation

Project

(RGAP)

v7

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(http://rice.plantbiology.msu.edu), respectively. The molecular weight (MW) and theoretical

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pI of proteins were predicted using ProtParam software (http://web.expasy.org/protparam).

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Thereafter, the neighbor-joining phylogenetic tree of MeDELLAs, AtDELLAs and OsSLR1

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was constructed using MEGA5.05 software and Clustalx 1.83 software (Tamura et al., 2011).

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Additionally, gene structure and conserved motif analyses of MeDELLAs were performed

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using Gene Structure Display Server (GSDS) v2.0 (http://gsds.cbi.pku.edu.cn/index.php) (Hu

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et

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(http://meme-suite.org/tools/meme), respectively.

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2.3. RNA isolation, reverse transcription and quantitative real-time PCR

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Total RNA was isolated using AxyPrepTM Multisource Total RNA Miniprep Kit

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(AYXGEN-09113KD1, Santa Clara, California, USA), purified using RNase-free DNase

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(NEB, M0303S, USA) to remove contaminating DNA, according to the manufacturer’s

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protocol. The quality and concentration of total RNA was detected using RNA

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electrophoresis and quantified using NANODROP 2000 (Thermo Scientific, Waltham,

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Massachusetts, USA). After verification, first-strand cDNA was synthesized using RevertAid

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First Strand cDNA Synthesis Kit (Thermo Scientific, K1622, Waltham, Massachusetts, USA).

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Thereafter, the diluted cDNA together with TransStart Tip Green qPCR SuperMix (TransGen

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Biotech, AQ141, Beijing, China) were used for quantitative real-time PCR in LightCycler®

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96 Real-Time PCR System (Roche, Basel, Switzerland), according to the manufacturer’s

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instruction. With the Ct values, all the transcript levels were quantified using the comparative

2015)

and

Multiple

Em

for

Motif

Elicitation

(MEME)

v4.11.0

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ACCEPTED MANUSCRIPT ∆∆Ct method in comparison to the reference gene MeEF1a. The primers were listed in

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Supplemental Table S1.

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2.4. Expression vector construction and transient expression in Nicotiana benthamiana

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leaves

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The CDS of MeDELLAs were amplified by PCR and cloned into the pEGAD vector (Cutler

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et al., 2000) by double enzyme digestion to construct recombinant plasmids

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35S::GFP-MeDELLAs. The primers were listed in Supplemental Table S2. After restriction

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enzyme identification and sequence analysis, the recombinant plasmids were transformed

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into Agrobacterium tumefaciens strain GV3101. About 4-week-old Nicotiana benthamiana

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leaves were syringe infiltrated of Agrobacterium tumefaciens harbouring different

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recombinant plasmids and P19 plasmid as Sparkes et al. (2006) described. At 2 days post

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infiltration

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4′,6-diamidino-2-phenylindole (DAPI) for 30 min to indicate cell nuclei, and further used for

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green fluorescent detection using a confocal laser-scanning microscope (TCS SP8, Leica,

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Heidelberg, Germany).

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2.5. Virus-induced gene silencing (VIGS) in cassava

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For the constructs of VIGS vectors, the partial CDS of MeDELLAs were amplified by PCR

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and cloned into the pTRV2 vector (Liu et al., 2002) by double enzyme digestion. The primers

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were listed in Supplemental Table S2. After restriction enzyme identification and sequence

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analysis, the recombinant plasmids of pTRV2-MeDELLAs and the pTRV1 plasmid were

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transformed into Agrobacterium tumefaciens strain GV3101. About 4-week-old cassava

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leaves were syringe infiltrated of Agrobacterium tumefaciens harbouring different

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recombinant plasmids of pTRV2-MeDELLAs or the pTRV1 plasmid as Wei et al. (2017)

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described. Further analyses of gene expression and disease resistance were performed in the

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leaves at 14 dpi.

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2.6. Bacterial Pathogen inoculation

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The bacterial pathogen of Xanthomonas axonopodis pv. manihotis Hainan (Xam Hn) was

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isolated from diseased cassava SC124 according to the Koch’s rule (Lopez et al., 2005, Wei

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et al., 2017). After streaked out on LB liquid culture at 28°C for 12 h, the bacterial culture of

the

infiltrated

leaves

were

incubated

with

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ACCEPTED MANUSCRIPT Xam was transferred to new LB liquid culture at 28°C to reach OD600 of about 0.6. Thereafter,

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the colony-forming units (cfu) per ml were quantified based on previous study (Wei et al.,

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2017), OD600 of 0.2 is about 108 cfu ml-1 for Xam. After diluted to 108 cfu ml-1 with 10 mM

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MgCl2, the bacterial culture with 0.05% silwet L-77 was syringe infiltrated into the abaxial

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side of plant leaves. At each indicated time-point, plant leaves were harvested for bacterial

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population assay as previously described (Wei et al., 2017).

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2.7. Determination of callose deposition

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Callose deposition in plant leaves was measured as Hauck et al. (2003) described. Briefly, the

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pathogen-infected plant leaves were stained in 0.01% (w/v) aniline blue solution for 1 h and

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mounted in 50% (v/v) glycerol. Then the callose depositions in plant leaves were visualized

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using fluorescence microscope (DM6000B, Leica, Heidelberg, Germany) and quantified by

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the ImageJ software.

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2.8. Statistical analysis

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At least three biological replicates were performed in each experiment, and the underlying

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average means and SDs were shown. After analysis using ANOVA and student t-test, the

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significant differences in comparison to mock (empty vector) were shown as asterisk symbols

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(*) at p<0.05.

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3. Results

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3.1. Comprehensive identification and analyses of MeDELLAs

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As shown in Supplemental Table S3, the detailed information of 4 MeDELLAs including the

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locus name and length of coding sequence were listed by identification and confirmation

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using CDD and Pfam softwares. The neighbor-joining phylogenetic tree of 4 MeDELLAs, 5

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AtDELLAs and OsSLR1 was constructed to provide insight into the evolution link. The

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phylogenetic analysis indicated a closer relationship between MeDELLAs and AtDELLAs

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(AtGAI, AtRGA, AtRGL1/2/3) (Fig. 1).

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Gene structure analysis found that all MeDELLAs have no intron in the genome (Fig. 2A).

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Conserved motif analysis using MEME v4.11.0 identified 10 enriched motifs, especially

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motif 6 (Fig. 2B). Both gene structure and conserved motif analyses confirmed the

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identification of 4 MeDELLAs. 7

ACCEPTED MANUSCRIPT 3.2. Expression profile of MeDELLAs in response to flg22 and Xam

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To investigate the possible in vivo roles of MeDELLAs, the expression profile of 4

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MeDELLAs in response to flg22 and Xanthomonas axonopodis pv manihotis (Xam) were

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analyzed by quantitative real-time PCR. We found that the transcript levels of 4 MeDELLAs

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were commonly up-regulated upon Xam treatment at 6 h post infiltration (hpi) although their

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expression differed at earlier stages (1 hpi and 3 hpi) (Fig. 3A-D). Upon flg22 treatment, the

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expression of 4 MeDELLAs was also commonly regulated (Fig. 3A-D). The changes of

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expression levels of 4 MeDELLAs upon flg22 and Xam treatment indicated the possible

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involvement of MeDELLAs in plant defense responses.

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3.3. Subcellular localization of MeDELLAs

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To analysis the subcellular location of MeDELLAs, the coding regions of MeDELLAs were

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fused to green fluorescent protein (GFP). After transient expression in Nicotiana

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benthamiana leaves, the green fluorescent of 4 MeDELLAs-GFP was visualized in both

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cytoplasm and cell nuclei as co-localized with DAPI-stained cell nuclei (Fig. 4).

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3.4. MeDELLAs positively regulate defense response to cassava bacterial blight

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To further reveal the in vivo roles of MeDELLAs in plant defense responses, we investigated

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disease resistance in plants with changed expression of these genes. Through overexpression

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in Nicotiana benthamiana, we found that MeDELLAs conferred improved disease resistance

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against cassava bacterial blight, as evidenced by less bacterial number in the plant leaves (Fig.

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5). Through VIGS in cassava (Wei et al., 2017), we found that MeDELLA1-, MeDELLA2-,

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MeDELLA3- and MeDELLA4-silenced plants exhibited decreased disease resistance, as

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evidenced by the resistance gene expression (Fig. 6A), cassava leaf growth (Fig. 6B) and

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bacterial proliferation (Fig. 6C). These results indicated that MeDELLAs are positive

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regulators of disease resistance against cassava bacterial blight.

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3.5. MeDELLAs regulate callose depositions and defense-related genes

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To further investigate the underlying mechanism of MeDELLAs-mediated defense response,

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we dissected the effect of MeDELLAs on pathogen associated molecular patterns

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(PAMPs)-triggered

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MeDELLA4-silenced plants through VIGS displayed less callose depositions (Fig. 7A) and

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lower transcript levels of defense-related genes (pathogensis-related genes (PRs) (Fig. 7B).

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immunity

(PTI).

MeDELLA1-,

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MeDELLA2-,

MeDELLA3-

and

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These results suggested the effect of MeDELLAs on PTI.

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4. Discussion

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All kinds of stresses (including pathogen infection and abiotic stress) are the major issues

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affecting and harming modern agricultural production. Thus, comprehensive investigation of

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plant stress responses and underlying molecular mechanisms, cultivation of stress resistant

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varieties through modern biological technology are of great importance all over the world

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(Achard et al., 2006, 2008a, b). As sessile organisms, most of plants except some aquatic

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plants are not removable, so plants have to response to harmful conditions. All stress signals

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are first perceived by the plant cell membrane receptors, and translated by secondary

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messengers to the downstream, resulting in activation of protein kinases, following

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modulation of gene expressions and protective responses (Yang et al., 2013a, b). Although the

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roles are different, all plant hormones including auxin, GA, SA, JA, ET, ABA, CK, BR and

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SL are involved in plant defense responses (Fu and Harberd, 2003; Yang et al., 2013a, b).

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Based on previous studies, AtDELLAs interact with multiple transcription factors of all these

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hormones, including ABI3/5 in ABA signaling, BZR1 in BR signaling, ARF6 in auxin

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signaling, EIN3 in ET signaling, JAZs and MYC2 in JA signaling, B-ARRs in CK signaling,

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D14 in SL signaling (Davière and Achard, 2016; Gao et al., 2011; Hou et al., 2010; Li et al.,

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2015, 2016; Lozano-Juste and León, 2011). The complex crosstalks of AtDELLA and plant

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hormones are consistent with the wide involvement of AtDELLA in plant development and

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stress responses (Davière and Achard, 2016). However, no DELLA has been identified in

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cassava, one of the most important tropical crops (Camilo et al., 2005; Muñoz-Bodnar et al.,

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2014).

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In this study, 4 MeDELLAs were genome-widely identified in cassava, as evidenced by

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the evolutionary tree, gene structures and motifs analyses. Gene expression analysis indicated

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that 4 MeDELLAs were commonly regulated by flg22 and Xam, indicating the possible

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involvement of them in plant defense response. Through overexpression in Nicotiana

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benthamiana and gene silencing by VIGS in cassava, we found that MeDELLAs positively

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regulated disease resistance against cassava bacterial blight. These results were in accordance

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with previous studies that AtDELLA proteins were also involved in plant response to bacteria 9

ACCEPTED MANUSCRIPT 1

and fungi (Tan et al., 2014) and OsSLR1 was a positive regulator of hemibiotroph

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resistance (Vleesschauwer et al., 2016). Additionally, the significant effects of MeDELLAs on

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callose-associated cell wall and defense-related genes (MePRs) indicated the positive role of

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MeDELLAs on PTI, which may be responsible for MeDELLAs-mediated defense resistance. Although the detailed underlying mechanism of MeDELLAs-mediated defense resistance

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was unclear, the present study provided strong evidence that 4 MeDELLAs are positive

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regulators of disease resistance against cassava bacterial blight. AtDELLAs interact with

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multiple transcription factors of plant hormones (Davière and Achard, 2016; Gao et al., 2011;

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Hou et al., 2010; Li et al., 2015, 2016; Lozano-Juste and León, 2011), in accordance with the

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wide involvement of AtDELLA in plant development and stress responses (Davière and

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Achard, 2016). Thus, the identification of MeDELLA-interacting proteins will provide more

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clues to the underlying mechanism in MeDELLAs-mediated defense response as well as

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other in vivo roles of MeDELLA.

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Taken together, this is the first study identifying MeDELLAs as positive regulators of disease resistance against cassava bacterial blight.

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Acknowledgements

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We thank Dr. Chris R. Somerville and Dr. Jie Zhou for sharing the vector plasmids. This

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research was supported by the National Natural Science Foundation of China (No.31760067),

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the startup funding and the scientific research foundation of Hainan University (No.kyqd1531)

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to Haitao Shi.

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Conflicts of Interest

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The authors declare that they have no conflicts of interest.

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Figure legends

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Figure 1. The neighbor-joining phylogenetic tree of 4 MeDELLAs, 5 AtDELLAs and

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OsSLR1.

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Figure 2. Gene structure and conserved motif analyses of MeDELLAs. (A) The gene

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structure analysis using GSDS v2.0. The CDS region and upstream/downstream were shown

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in red and blue colors, respectively. (B) The conserved motif analysis using MEME v4.11.0.

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The sequences of enriched 10 motifs were shown.

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Figure 3. The transcript levels of MeDELLA1 (A), MeDELLA2 (B), MeDELLA3 (C)

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MeDELLA4 (D) in response to flg22 and Xam treatments. About 30-day-old cassava

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leaves were sprayed with water (mock for flg22 treatment), or 10 µM flg22, syringe

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infiltrated by 10 mM MgCl2 or 108 cfu ml-1 of Xam with 10 mM MgCl2, and leaves samples

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were harvest for gene expression analysis at indicated time-points. The transcript levels were

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shown in relative to the mock treatment. The significant differences in comparison to mock

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were shown as asterisk symbols (*) as p<0.05.

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Figure 4. Subcellular localization of MeDELLAs in Nicotiana benthamiana leaves. The

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Agrobacterium tumefaciens harbouring different recombinant plasmids and P19 plasmid were

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syringe infiltrated into Nicotiana benthamiana leaves. At 2 days post infiltration, the green 14

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fluorescent and DAPI-stained cell nuclei were visualized by confocal laser-scanning

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microscope. Bars = 25 µm.

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Figure 5. Transient expression of MeDELLAs confers disease resistance. The

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Agrobacterium tumefaciens harbouring different recombinant plasmids and P19 plasmid were

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infiltrated into Nicotiana benthamiana leaves. At 2 dpi, the Nicotiana benthamiana leaves

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were syringe infiltrated by 108 cfu ml-1 of Xam for additional 0, 2, 4, 6 days, and the bacterial

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number in the leaves were assayed at indicated time-points. At least 15 leaves were assayed

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in each biological repeat, and at least three biological repeats were performed for each data.

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The significant differences in comparison to vector transformation were shown as asterisk

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symbols (*) as p<0.05.

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Figure 6. MeDELLA-silenced plants exhibited decreased disease resistance. (A) The

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transcript levels of indicated genes in the MeDELLA1-, MeDELLA2-, MeDELLA3- and

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MeDELLA4-silenced plants. (B) The phenotype of MeDELLA-silenced plants in response to

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cassava bacterial blight. (C) The bacterial number of MeDELLA-silenced plants in response

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to cassava bacterial blight. For the assay, the Agrobacterium tumefaciens harbouring

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pTRV2-MeDELLAs and the pTRV1 plasmid were syringe infiltrated cassava leaves. At 14

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dpi, the cassava leaves were infected by 108 cfu ml-1 of Xam for additional 0, 2, 4, 6 days, and

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then the bacterial number in the leaves were assayed at indicated time-points. At least 15

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leaves were assayed in each biological repeat, and at least three biological repeats were

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performed for each data. The significant differences in comparison to vector transformation

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were shown as asterisk symbols (*) as p<0.05.

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Figure 7. MeDELLA-silenced plants regulate PTI. (A) The visualization and quantification

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of callose in MeDELLA-silenced plant leaves. White dots indicate callose depositions, and the

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relative average means and SDs were shown in the figures. Bar = 500 µm. (B) The transcript

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levels of MePRs in MeDELLA-silenced plant leaves. Significant differences were shown as

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asterisk symbols (*) at p<0.05.

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Table S1. The primers used in the quantitative real time-PCR.

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Table S2. The primers used in the vector construction.

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Table S3. Identification of 4 DELLA proteins in cassava.

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1. MeDELLAs are commonly regulated by flg22 and Xam. 2. MeDELLAs confer improved defense response.

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3. MeDELLAs are essential for defense response in cassava.