Of pests and sugarcane…

(Functional´omics of the ratoon stunting disease of sugarcane)

Luis E. Aranha Camargo Dept.Fitopatologia e Nematologia - ESALQ

Projects on biotic stress 1) Optimization of the biocontrol of the sugarcane borer (Diatraea saccharalis) by Trichograma galloi through mathematical modelling.

Marat Rafikov – Federal University of the ABC region

2) Herbivory and intracellular transport of proteins

Marcio de Castro e Silva Filho – University of São Paulo 3) Genetics and genomics of Sporisorium scitaminae, the causal agent of sugarcane smut.

Claudia Barros Monteiro-Vitorello – University of São Paulo 4) Functional´omics of the ratoon stunting disease of sugarcane

Luis E. Aranha Camargo – University of São Paulo

Projects on biotic stress 1) Optimization of the biocontrol of the sugarcane borer (Diatraea saccharalis) by Trichograma galloi through mathematical modelling.

Marat Rafikov – Federal University of the ABC region

2) Herbivory and intracellular transport of proteins

Marcio de Castro e Silva Filho – University of São Paulo 3) Genetics and genomics of Sporisorium scitaminae, the causal agent of sugarcane smut.

Claudia Barros Monteiro-Vitorello – University of São Paulo 4) Functional´omics of the ratoon stunting disease of sugarcane

Luis E. Aranha Camargo – University of São Paulo

Mathematical modelling of the biocontrol of Diatraea saccharalis Marat Rafikov

OBJECTIVE:

-Develop a mathematical model to predict the control efficacy of the sugarcane borer in response to the number of parasitoids released in the

field.

How many parasitoids should be released in order to maintain the borer population levels just below the economical threshold (ET)?

ET = the maximum number of pest insects above which economic losses occur

Parasitoid (Trichograma galloi)

Borer (Diatraea)

Population levels of pest over time in response to relasing different Numbers of parasitoids/hectare

ET

No control

ET

3,500 parasitoids/ha

ET

2,500 parasitoids/ha

ET

1,500 parasitoids/ha

ET = 2,500 larvae/ha

Projects on biotic stress 1) Optimization of the biocontrol of the sugarcane borer (Diatraea saccharalis) by Trichograma galloi through mathematical modelling.

Marat Rafikov – Federal University of the ABC region

2) Herbivory and intracellular transport of proteins

Marcio de Castro e Silva Filho – University of São Paulo 3) Genetics and genomics of Sporisorium scitaminae, the causal agent of sugarcane smut.

Claudia Barros Monteiro-Vitorello – University of São Paulo 4) Functional´omics of the ratoon stunting disease of sugarcane

Luis E. Aranha Camargo – University of São Paulo

• Characterize the response of sugarcane to major insect pests;

• Understand the molecular adaptive mechanisms of sugarcane pests

• High-throughput gene expression analyses of sugarcane proteinase inhibitors (PI) and serine-proteinases (SP) in response to herbivory by the sugarcane borer (Diatraea saccharalis);

• Characterize the molecular mechanisms involved in subcellular protein localization in plant cells .

Herbivory and intracellular transport of proteins Marcio de Castro Silva Filho

OBJECTIVES:

-Sugarwin – homologues of barley wound-inducible protein -Induction occurs in response to wounding and borer attack -Has no effect on insect but is fungitoxic to Fusarium! -Concerted plant response to the anticipated invasion by the fungus

apop Co

ntr

ol -

PB

S CFW DI

C

SUG

AR

WI

N2

16

0

µM

Induced apoptosis

Rapid alkalinization factor (RALF) is part of a growing family of small peptides with hormone characteristics in plants.

Four copies of SacRALF genes were found in sugarcane plants All four SacRALF genes are highly expressed in young and expanding leaves and show a low or undetectable level of expression in expanded leaves The findings are consistent with SacRALF genes playing a role in plant development potentially regulating tissue expansion

Projects on biotic stress 1) Optimization of the biocontrol of the sugarcane borer (Diatraea saccharalis) by Trichograma galloi through mathematical modelling.

Marat Rafikov – Federal University of the ABC region

2) Herbivory and intracellular transport of proteins

Marcio de Castro e Silva Filho – University of São Paulo 3) Genetics and genomics of Sporisorium scitaminae, the causal agent of sugarcane smut.

Claudia Barros Monteiro-Vitorello – University of São Paulo 4) Functional´omics of the ratoon stunting disease of sugarcane

Luis E. Aranha Camargo – University of São Paulo

OBJECTIVES:

Evaluate the genetic and pathogenic variability of S. scitaminae in Brazil.

Sequence the genome of S. scitaminae and determine the chromosome number and size.

Transcriptomics of the host-pathogen interaction based on RNAseq;

Develop a transformation protocol (Agrotransformation) for S. scitaminae with GFP and dsRed to monitor the infection process.

Genetics and genomics of Sporisorium scitaminae Claudia Barros Monteiro-Vitorello

Genetic variability: new markers (telomeric sequences) unveiled higher levels of genetic varialibilty than reported before

AFLP

RFLP (telomeres)

Genome sequencing: determination of chromosome number by PFGE

and BAC-based sequencing

Agrotransformation of Sporisorium for monitoring plant infection

Co-cultivation of S. scitamineum and A. tumefaciens EHA105

transformed

wild-type

Projects on biotic stress 1) Optimization of the biocontrol of the sugarcane borer (Diatraea saccharalis) by Trichograma galloi through mathematical modelling.

Marat Rafikov – Federal University of the ABC region

2) Herbivory and intracellular transport of proteins

Marcio de Castro e Silva Filho – University of São Paulo 3) Genetics and genomics of Sporisorium scitaminae, the causal agent of sugarcane smut.

Claudia Barros Monteiro-Vitorello – University of São Paulo 4) Functional´omics of the ratoon stunting disease of sugarcane

Luis E. Aranha Camargo – University of São Paulo

Functional´omics of the ratoon stunting disease Luis E. Aranha Camargo

OBJECTIVES:

- Identify sugarcane genes and proteins differentially expressed in a susceptible cultivar during infection with Lxx based on microarray hybridizations and 2-D DIGE coupled with protein sequencing by LC-MS/MS.

- what are the metabolic pathways responsive to Lxx infection?

- can plant stunting be explained by proteomic/transcriptomic data?

- Develop a bioassay to characterize a putative toxin secreted by Lxx

RSD symptoms: shortening of internodes leads to loss of biomass

Left: stunted cane Right: healthy cane Susceptible variety

with and without symptoms

Experimental design Treatments: •susceptible variety SP80-3280 • inoculated or mock-inoculated with Lxx • 30 and 60 days after inoculation (DAI) •3 biological replicates

T0 (time zero)

-Proteomics -2D-DIGE

30 DAI 60 DAI

Comparisons: Mock-inoculated 30 x 60

Inoculated 30 x 60

- Transcriptomics (SUCEST agilent chip)

Comparisons: Mock-inoculated x inoculated

(30 and 60 DAI)

Results

Taken together, results of proteomics and trancriptomics indicated that three major categories

are differentially regulated during colonization by Lxx:

Cell cycle

Hormone synthesis

Signal transduction

Strikingly, genes and proteins involved in cell cycle were all repressed during colonization

S

G1

G2

M CDK2 CDK2

Aur kinase

TPXKlp2

CDC7 MCM2-7

H4

CHD1 DNA topoisomerase

Kif4

KIF22

Tangled1 KNOLLE

H2A

SMC3 SMC2

CYCA1;1 CYCB2

CYC1B

MAD2

MAD3

CAF 1

Phi-1

up regulation of genes involved in hormone synthesis...

Transcript_name Hormone Gene Ontology 60 DAI

SCCCLR1022A02.g Ethylene 1-aminocyclopropane-1-carboxylate

oxidase 5,59

SCSFRT2070A10.g Ethylene 2OG-Fe oxygenase family 1,98

SCQGAM2028D11.g Ethylene 1-aminocyclopropane-1-carboxylate

synthase 4,91

SCCCCL3002B05.b Auxin Indole-3-acetic acid-amido synthetase 3,02

SCSBAM1085B06.g Jasmonic acid Omega-3 fatty acid desaturase 2.07

Transcript_name Function_category Gene Ontology 60

DAI

SCVPFL3046E09.b Signal Transduction Acid phosphatase 2,33

SCMCRT2108E08.g Signal Transduction putative RLCK 2,37

SCEPAM1018C05.g Signal Transduction Transcription factor PCF7 -2,04

SCBGLR1115B08.g Signal Transduction Receptor Ser/Thr kinase 1,99

SCCCLR1075G12.g Signal Transduction Receptor Ser/Thr kinase-unclassified 2,92

SCQGST1032D08.g Signal Transduction MYB 2,44

SCCCCL4002B07.g Signal Transduction Ethylene-responsive factor-like protein 1 3,15

SCJFRT1060C03.g Signal Transduction Putative ribonuclease H protein 3,44

SCCCRZ1004G12.g Signal Transduction Putative NAC domain-containing protein 2,30

SCCCRT1001B08.g Signal Transduction Receptor Ser/Thr kinase-unclassified 2,97

SCCCCL4005C09.g Signal Transduction Light-inducible protein CPRF2 2,28

SCCCLR2C02G01.g Signal Transduction Calcium dependent protein kinase 2,27

SCCCCL4001A01.g Signal Transduction Probable WRKY transcription factor 51 3,38

SCCCLR2004B09.g Signal Transduction Transcription factor bHLH96 2,49

SCEQLB2020F07.g Signal Transduction Protein kinase unclassified 3,13

SCJFRZ2015H10.g Signal Transduction Lectin-like receptor kinase-like protein 2,61

SCAGLR1021G10.g Signal Transduction KNOTTED1 homeodomain protein 4,62

SCACLR1130H08.g Signal Transduction Zinc finger protein -3,55

SCCCAD1003G07.g Signal Transduction Homeobox-leucine zipper protein ROC7 -1,87

SCJFLR1013D05.g Signal Transduction Homeobox-leucine zipper protein ROC1 -1,90

SCCCCL3120A10.b Signal Transduction Auxin response factor 12 2,30

regulation of genes and proteins involved in signal transduction

- 1) Down regulation of genes involved in cell cycle is consistent with the main symptom of the disease- stunting (generalized poor development)

- 2) Cell cycle arrest might result from modulation of hormone signalling.

- Interesting hypothesis: modulator is secreted by Lxx (effector?)

Conclusions : proteomics and transcriptomics

Wrap-up: validation of candidate genes and hormone profiling

Lxx

Cell Cycle Hormones Signal transduction

Characterization of a putative toxin secreted by Lxx

Organic extracts of Lxx cultures inhibit the germination of lettuce seeds

-Toxin production is dependent on osmotic stress (PEG) and is inhibited to some degree by fosmidomycin - Since fosmidomycin inhibits the synthesis of carotenoids in prokaryotes, it is assumed that the toxin has a carotenoid precursor

-Hypotheses: toxin is an analog of ABA and therefore arrests cell cycle and plant growth (?)

Lxx + PEG medium + PEG Lxx + PEG + Fos Medium + PEG + Fos

Thank you! (leacamar@usp.br)