Reniform Nematode (<I>Rotylenchulus

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12-2014 Reniform nematode (Rotylenchulus reniformis) manipulation of host root gene expression during syncytium formation in upland cotton (Gossypium hirsutum) Wei Li Clemson University, [email protected]

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Recommended Citation Li, Wei, "Reniform nematode (Rotylenchulus reniformis) manipulation of host root gene expression during syncytium formation in upland cotton (Gossypium hirsutum)" (2014). All Theses. 2038. https://tigerprints.clemson.edu/all_theses/2038

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RENIFORM NEMATODE (ROTYLENCHULUS RENIFORMIS) MANIPULATION OF HOST ROOT GENE EXPRESSION DURING SYNCYTIUM FORMATION IN UPLAND COTTON (GOSSYPIUM HIRSUTUM)

A Thesis Presented to the Graduate School of Clemson University

In Partial Fulfillment of the Requirements for the Degree Master of Science Plant and Environmental Sciences

by Wei Li December 2014

Accepted by Dr. Paula Agudelo, Committee Co-chair Dr. Christina Wells, Committee Co-chair Dr. Patrick Gerard

Abstract

Background: The semi-endoparasitic reniform nematode (Rotylenchulus reniformis) is a major yield-limiting pest of multiple crops in the tropics and sub-tropics, including upland cotton

(Gossypium hirsutum). Reniform-resistant cotton varieties are urgently needed, but genes that confer resistance to reniform nematode have not been identified in any species. Parasitism by reniform nematode involves significant developmental changes in plant roots, leading to the formation of multicellular feeding structures called syncytia. Here, we present de novo transcriptomes assembled from syncytial and non-syncytial cotton roots on three sampling dates across a 12-day time course.

Results: Total mRNA samples extracted from reniform-infected and uninfected G. hirsutum roots were sequenced on the Illumina HiSeq 2000 platform, generating over 400 million paired-end reads. A de novo root transcriptome for G. hirsutum was assembled with the Trinity pipeline, generating 156,156 unique contigs with an N50 of 712 bp. After removal of contigs from nematodes and other soil organisms, reads from 18 individual RNA samples were mapped back to the cotton reference transcriptome to quantify gene expression at 3, 9 and 12 days after inoculation (DAI). Overall, 432, 266 and 229 genes were significantly up-regulated and 297, 325, and 232 genes were significantly down-regulated in infected vs. uninfected roots at 3, 9, and 12

DAI, respectively (FDR = 0.05). GO-enrichment analyses identified 48 gene ontology terms that were significantly enriched in the set of 1500 genes differentially expressed on at least one sampling date. These included cell periphery, membrane and plasma membrane, catalytic activity, oxidoreductase activity, and response to stimulus and stress. Five genes were significantly up-regulated in syncytial roots across all sampling dates: an ABC transporter, a cytochrome p450, glutathione s-transferase, a homolog of the multi-drug and toxic compound

ii extrusion gene transparent testa 12, and a bel1 transcription factor. Twenty-eight genes were significantly down-regulated across all sampling dates, many of which were involved in cell wall processes and plant defense.

Conclusions: Comprehensive gene expression profiles of syncytial development significantly advance our current understanding of plant resistance to reniform nematode. In future work, we will use these expression data to construct gene network models of syncytium formation, with the long-term goal of disrupting the networks that underlie successful nematode infection.

Gaining new insights into the mechanisms of plant response to reniform nematode has practical significance for nematode control through the development of resistant crop varieties.

Keywords: Rotylenchulus reniformis, Gossypium hirsutum, Transcriptome, Differential expression

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ACKNOWLEDGEMENTS

I would never have been able to finish my thesis without the guidance of my committee members, help from friends, and support from my family.

I would like to express my deepest gratitude to my advisors, Dr. Paula Agudelo and Dr.

Christina Wells for providing me the opportunity to complete my Master degree. Their excellent guidance, caring, patience and timely advice helped me to finish this project. It has been a great honor to have them as my major advisors.

I am thankful to my committee member, Dr. Patrick Gerard, who guided, encouraged and supported me for the past two and half years and helped me build up my background in statistics.

I would like to thank Ms Nancy Korn in histology core facility, who trained me patiently with various sectioning methods.

I want to thank Dr. Bielenberg and Dr. Saara DeWalt from Department of Biological Sciences for the opportunity to be a Teaching Assistant for the courses Plant Physiology and Biology of

Plants, respectively. Both were excellent experiences that I really enjoyed.

I would like to thank all the past and current members of Clemson University Nematology laboratory. I will always be proud to be associated with Clemson University and a lab directed by

Dr. Paula Agudelo.

I feel thankful to the faculty, staff, and students of Plant and Environmental Sciences Program.

Finally, I would like to thank my parents, whose unconditional love and support gave me the spirit to accomplish all my work for my Master degree. I want to offer my regards and blessings to all those who supported me while I was working at Clemson University.

TABLE OF CONTENTS Page TITILE PAGE ...... i ABSTRACT ...... ii ACKNOWLEDGEMENTS ...... iv LIST OF TABLES ...... vi LIST OF FIGURES ...... vii BACKGROUND ...... 1 MATERIALS AND METHODS...... 3 RESULTS ...... 7 DISCUSSION ...... 11 APPENDICES ...... 36 REFERENCES ...... 43

LIST OF TABLES

Table Page

1. Summary of sequencing and assembly ...... 20

2. Top twenty up- and down-regulated genes on each sampling date. Sequence descriptions are based on top BLASTx hits for each contig. Normalized counts are expressed as number of reads mapping to the contig per million mapped reads (N=3 for each date x infection status combination). * means the same or similar contigs show multiple times in same subtable ...... 21

3. Thirty-three genes differentially expressed in syncytial roots across all sampling dates ...... 27

LIST OF FIGURES

Figure Page

1. Split-root system. Split root system into two different pots with the help of Y-shape (A and B) tube. Inoculate half the root system with reniform nematodes (3000 reniform nematodes/pot) (C). RNA extraction from inoculated and noninoculated root tissues can be achieved from the same plant to separate local and systemic gene expression profiles...... 6

2. Resin section of reniform nematode penetration and syncytium formation. Syncytium cells were circled in red, and non-syncytium cells in yellow. DAI: days after inoculation; Ne: reniform nematode; SC: syncytium cells (circled in red line) ...... 29

3. Unique contigs annotation results from BLAST2GO. Contigs with top BLASTed plant species were regarded as putative cotton contigs, contigs with top BLASTed animal species were regarded as putative animal contigs, and other contigs include all top BLAST hits assigned as fungi, bacteria, and virus and so on ...... 30

4. DE contigs distribution on different dates. Pink bars present the significantly up-regulated contigs, and blue bars present the significantly down-regulated contigs ...... 31

5. Distribution of expression changes on different dates. Black dots show non-DE contigs, colored dots show all the DE contigs significant regulated with log2fold of 2 or greater presented as green ...... 32

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6. Common DE contigs shared between different dates. Date-specific contig expression profiling with List of Figures (Continued) Figure Page

Venn Diagram with each section showing the contig expression number ...... 33

7. GO annotation of DE unique contigs by BLAST2GO. The x-axis indicated the sub-categories and the y-axis indicated the number of unique contigs, and the unique contig number assigned with same GO terms were indicated on the top of each group (GO terms with more than 5 presented here). A for biological process, B for cellular component, and C for molecular function ...... 34

8. DE transcript factors ...... 35

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BACKGROUND

The semi-endoparasitic reniform nematode (Rotylenchulus reniformis) is a major yield-limiting pest of multiple agriculture crops in the tropics and sub-tropics [1, 2]. Its geographic range has expanded over the last 20 years, making it the most damaging pest of upland cotton (Gossypium hirsutum) across much of the southeastern United States [3]. All commercial cultivars of G. hirsutum are currently susceptible to reniform nematode [4-6]. Management options for its control are limited [4, 7-10], and the most effective chemical controls (e.g. methyl bromide and aldicarb) have been withdrawn from the market due to their detrimental effects on the environment [11, 12]. Resistant cotton varieties are urgently needed, but genes that confer plant resistance to reniform nematode have not been identified.

Multiple aspects of R. reniformis biology contribute to its establishment as a significant plant pathogen [1, 13]: it has a broad host range among both dicots and monocots, reducing options for control by crop rotation [8, 14]; it has the ability to enter an anhydrobiotic state and remain viable in dry soil for long periods, facilitating its survival in the absence of a host [15]; and it possesses a high reproductive rate, short life cycle, and the ability to colonize the soil from depths below the effective range of nematicide treatment [2].

Parasitism by reniform nematode involves significant developmental and physiological changes in plant root cells, leading to the formation of specialized feeding structures called syncytia. After penetrating the root, an infective immature female will insert her stylet into an endodermal cell, releasing a proteinaceous secretion that triggers syncytium formation through cell wall dissolution and cytoplasmic coalescence of adjacent pericycle cells [13, 16, 17]. The identity of the secreted reniform effectors and the specific plant gene networks they influence are unknown [18, 19]. Formation of similar feeding structures by cyst nematodes appears to

1 require the suppression of plant defenses and the manipulation of genes involved in cell death, cell cycle control and cell wall modification [20-26].

Here we describe the de novo assembly of a G. hirsutum transcriptome and present a time course of gene expression during the formation of reniform syncytia in cotton roots. We describe a split-root growth system that allows us to separate local gene expression changes in syncytial roots from systemic changes in gene expression. GO term enrichment analyses highlights categories of host plant genes that represent potential targets for breeding or genetic engineering of reniform-resistant cotton cultivars.

MATERIALS AND METHODS

Plant growth and nematode inoculation – Surface-sterilized seeds of G. hirsutum cv.

Deltapine 50 were pre-germinated on moist paper towels in a growth room maintained at 28 ±

2C with a 14-h light/10-h dark photoperiod. After cotyledon emergence, 20 similar-sized, healthy seedlings were transferred to vermiculite and their root tips pruned to encourage lateral root proliferation. After one week, seedlings were transplanted into adjacent 300-cm3 pots containing pasteurized fine sand, splitting their root systems equally between the two pots by means of a Y-shaped plastic tube (Figure 1). This split-root growth system was designed to permit

RNA isolation from infected and uninfected roots of the same plant, effectively eliminating gene expression differences associated with systemic nematode responses and highlighting those differences associated specifically with syncytium formation.

A reniform nematode suspension was prepared by centrifugal sugar flotation from infested cotton field soil collected in St. Matthews, SC [27]. Reniform nematodes were collected on a 35 uM sieve and resuspended in tap water. One week after transplant to the split-root system, one pot per plant was randomly selected for inoculation with 2 ml of nematode suspension (approx.

3,000 nematodes), while the other pot received an equal volume of tap water. Plants were maintained in the growth room for 12 days after inoculation, receiving 20 ml of water every day until harvest.

Microscopy – Three replicate plants were harvested at 3, 9 and 12 days after inoculation (DAI) to assess the histopathology of syncytium development. Five to ten pieces of infected and uninfected root tissue from each plant were fixed overnight in freshly prepared 3.7%

Formaldehyde/Acetic Acid (FAA: 50% ethanol, 5% glacial acetic acid, 3.7% formaldehyde), dehydrated in graded ethanol (50%, 75%, 90-95%, 100% v/v), cleared in xylene, infiltrated in

3 ethanol, infiltrated in catalyzed resin, and finally embedded in resin (Spurr Low-Viscosity

Embedding Kit, Sigma-Aldrich, Inc). Embedded sample blocks were sectioned at 0.5 μm and stained with Azure II (1% in 1% sodium borate) and basic fuchsin (1% in 50% ethanol, diluted to

1/20 for use). Slides were viewed at 20-40X with an OLYMPUS Microscope BH-2 (OLYMPUS,

Japan), and images were captured using the ProgRes® microscope camera with ProgRes®

CapturePro image capture software (JENOPTIK, Germany). Syncytia appeared as fused, hypertrophied pericycle cells with dense cytoplasm and an increased number and size of organelles.

Transcriptome Sequencing – Approximately 500 mg (FW) of root tissue was harvested from both infected and the uninfected root systems of three replicate plants at 3, 9, and 12 DAI, for a total of 18 samples (2 infection statuses X 3 dates X 3 biological replicates). Tissue was preserved in RNAlater (QIAGEN, Valencia, CA) and immediately shipped to the University of Arizona

Genetics Core (Tucson, AZ) for 100-bp paired-end cDNA library preparation, barcode tagging, and sequencing on three lanes of the Illumina Hi-Seq platform (Illumina, San Diego, CA). Over 448 million paired-end 100-bp reads were generated, with an average of 24.9 million reads per sample. Read quality was assessed with FastQC [28], followed by adaptor trimming with

Trimmomatic [29] and content-dependent quality trimming with ConDeTri [30]. The average per-read PHRED quality score after trimming and filtering was 37.

Transcriptome Assembly and Annotation – Trimmed and filtered reads from all samples were combined for transcriptome assembly on the Texas Advanced Computer Center Stampede high performance computing cluster using the Trinity pipeline with default parameters [31]. The assembly generated 213,984 total sequences: 156,156 unique contigs and 57,828 alternate splice forms of these contigs. Functional annotation of the unique contigs was performed using

BLAST2GO [32], which executes a BLASTx search against the NCBI nonredundant database and assigns gene ontology (GO) terms, Interpro IDs, enzyme codes and Kegg pathways to sequences with a significant BLASTx hit (E-value < 1.0 e-6).

RNA for transcriptome assembly was isolated from nematode-infected and uninfected cotton roots growing in a non-sterile soil matrix. The resulting assembly therefore contained both cotton and nematode genes, as well as genes from fungal and microbial components of the rhizosphere. Putative cotton protein-coding genes were separated from other contigs prior to differential gene expression analysis. Two criteria were used to identify protein-coding cotton genes: (1) the contig had a significant BLASTx against the NCBI nr database, and (2) its top BLAST hit species was a plant. This approach may have discarded cotton genes with low sequence similarity to other known plant genes, as well as long non-protein coding RNAs.

Gene expression was quantified by mapping reads from individual RNA samples back to the cotton protein-coding gene transcriptome using RSEM [33]. Contigs with uniform low expression

(fewer than ten mapped reads across all samples) were filtered, and count data from the remaining genes were imported to DESeq2 for differential expression analysis using default settings [34]. Data were analyzed separately by date with treatment as the single model covariate, and principal components analysis (PCA) confirmed that each date’s samples could clearly be separated by inoculation status along at least one principal component

(Supplementary Figure 1). Significant differentially expressed contigs were identified based on a false discovery rate of 0.05.

A B C

Figure 1. Split-root system. Split root system into two different pots with the help of Y-shape (A and B) tube. Inoculate half the root system with reniform nematodes (3000 reniform nematodes/pot) (C).

RNA extraction from inoculated and noninoculated root tissues can be achieved from the same plant to separate local and systemic gene expression profiles.

RESULTS

Microscopy -- Resin sections prepared from inoculated roots at 3, 9 and 12 days after inoculation (DAI) documented the progress of syncytium formation through time (Figure 2). At 3

DAI, reniform nematodes had begun to penetrate intracellularly, with majority of samples showing a vermiform female nematode among the root cortical cells. In some samples, females had reached the initial endodermal cell, but no syncytia were visible. At 9 DAI, reniform nematodes had arrived at the endodermis and their stylets were visibly embedded in single endodermal cells. Developing syncytia were clearly visible as regions of enlarged, interconnected pericycle cells with disorganized cytoplasm. The dense cytoplasm of syncytial cells did not fill up all the cell space, but was surrounding a clear irregular area in the cell center instead. At 12 DAI, syncytia consisted of multiple layers of cells with numerous, enlarged organelles. Evenly distributed dense cytoplasm, enlarged nuclei and cell wall lysis in different stages are also readily observable.

Transcriptome assembly and annotation – RNA was extracted from inoculated and non-inoculated root tissue at 3, 9 and 12 DAI for transcriptome assembly and differential gene expression analysis. Illumina sequencing generated over 448 million raw reads with a mean length of 100 bp and an average GC content of 50% (Table 1). After filtering and trimming, 414 million clean reads from all samples were combined for de novo assembly with the Trinity pipeline, generating 156,156 unique contigs and 57,828 alternate splice forms of these contigs.

Mean contig length was 546 ± 602 bp, and contig N50 was 712 bp. Contigs were functionally annotated using BLAST2GO, which implements a BLASTx search against the NCBI non-redundant database and assigns top BLAST hit species, sequence descriptions and GO terms to contigs with significant hits (E-value < 1.0 e-6). Seventy percent of the assembled contigs were annotated with

7 at least one BLASTx hit (Figure 3).

Cotton protein-coding genes were separated from other contigs based on their top BLAST hit species prior to differential expression analysis (see Methods). Forty-four percent of

BLASTx-annotated contigs had a plant as their top BLAST hit species. By far the most common top BLAST hit species among plant contigs was Theobroma cacao (27,812 unique contigs), the only fully-sequenced species in the family Malvaceae to which cotton also belongs. Thirty-two percent of the annotated contigs had an animal as their top BLAST hit species. The nematodes

Ascaris suum and Caenorhabditis genus, as well as the common soil protozoa Acanthamoeba castellanii str. Neff, were the most common top BLAST hit species among animal contigs. The remaining 31% of annotated contigs included sequences whose top BLAST hit species were fungi, bacteria and viruses. The full transcriptome annotation with top BLAST hit species for each contig is provided in Supplementary File 1 (http://tinyurl.com/kkjc5b3).

Differential expression analysis – Genes differentially expressed between infected and non-infected root tissue were identified by mapping reads from individual samples back to the cotton protein-coding gene set using RSEM and DESeq2 (see Methods). Because of the split-root sampling scheme we employed, differentially expressed genes were unlikely to include genes whose expression was altered systemically throughout the plant in response to reniform feeding.

Rather, they represented genes whose expression was locally altered in syncytial roots.

There were 432, 266 and 229 significantly up-regulated genes and 297, 325, and 232 significantly down-regulated genes in syncytial roots compared to control roots at 3, 9, and 12

DAI, respectively (Figure 4). Twenty, 23, and 2 percent of these differentially-expressed genes had an absolute log2fold change of 2 or greater in syncytial roots at 3, 9, and 12 DAI (Figure 5).

The most highly up- and down-regulated genes on each date are given in Table 2. Normalized

8 mapped read counts for protein coding cotton genes in all samples are provided in

Supplementary File 2 (http://tinyurl.com/llutj3v). Thirty-three genes were differentially expressed in syncytial roots on all three sampling dates: 5 up-regulated and 28 down-regulated (Figure 6,

Table 3).

Fisher’s exact test was used to identify gene ontology (GO) terms that were enriched in the set of genes differentially expressed on at least one date (Figure 7). The majority of enriched cellular component GO terms were related to the cell wall, plasma membrane and extracellular space.

Enriched molecular function GO terms included catalytic activity, oxidoreductase activity, transcription factor activity and a number of terms related to carbohydrate metabolism, lipid binding and transport. Enriched biological process GO terms included those associated with carbohydrate and amino acid metabolism and transport, cell wall organization, localization, and response to stress and stimuli.

Differentially Expressed Transcription Factors – Of particular interest were differentially-expressed transcription factors, which are likely to be high level regulators of the syncytium developmental process. Ten percent (149) of the genes differentially expressed on at least one date in our study were transcription factors (Figure 8), including a large number of zinc-finger, NAC, ERF, WRKY and bHLH transcription factors. The majority (129) of these transcription factors were differentially expressed on all three time points, indicating that their up- or down-regulation was maintained throughout syncytium initiation and development.

However, four transcription factors were only differentially expressed only on date 3: two NAC transcription factors, a C2H2 zinc finger protein, and a myb transcription factor with high sequence similarity to ODORANT1 from Malus x domestica. Among the transcription factors whose log2fold change was 2 or greater were several NAC, ERF (ethylene-responsive ), bHLH,

LUX, WRKY and MYB transcription factors.

DISCUSSION

Overall Results – Infection by R. reniformis triggers numerous changes in the gene expression of cotton roots, ultimately redesigning root morphology to form permanent feeding sites

(syncytia). Previous studies have separated syncytium development into two stages: an initial stage characterized by partial cell wall lysis and separation (3-6 DAI), and a later anabolic stage characterized by organelle proliferation and the formation of secondary wall deposits (9-12 DAI)

[16, 17, 35].

In the present work, reniform nematodes had begun to penetrate the host roots intracellularly at 3 DAI. Syncytium development was initiated between 3 and 9 DAI and syncytia were mature by 12 DAI. In the initial stage (3DAI), reniform nematode were mainly observed in the root cortex, moving between cortical cells in a radial and/or vertical orientation. Nematodes stopped penetrating when they reached the endodermis and inserted their stylet into a single endodermal cell (the initial syncytial cell). Subsequent development of the syncytium took place within the pericycle, a single meristematic cell layer located just inside the endodermis.

In the anabolic stage (9 and 12 DAI), densely staining cytoplasm was pushed to boundary of interconnecting syncytial cells by an irregularly-shaped clear central vacuole. Enlarged nuclei and nucleoli were readily observed, surrounding by an increased number and size of cellular organelles. As noted in previous studies, cortical cells outside of the enlarging syncytium were compressed in some samples [16]. Previous work suggests that dense syncytial cytoplasm includes many small vacuoles, mitochondria, dictyosomes, rough endoplasmic reticulum and ribosomes [16, 17]. Increased numbers of organelles are thought to support the continuous polysaccharide and protein synthesis required to support the parasitizing nematode [16, 35].

Our 3 DAI samples captured the earliest transcriptome events in the reniform-cotton root

11 interaction that preceded penetration of the endodermis. Interestingly, the largest number of differentially expressed genes were observed at 3 DAI, prior to syncytium initiation. The majority of these genes were up-regulated, and it is tempting to speculate that they represent the beginnings of a plant pathogen defense program which is subsequently shut down by the infecting nematode. Supporting this interpretation is the observation that 39 up-regulated genes on 3 DAI were annotated with defense-related GO terms, compared to 13 and 11 genes on dates

9 and 12.

The initial (3 DAI) and anabolic (9 and 12 DAI) infection stages were both histologically and transcriptomically distinct (Figure 6). Date 9 and 12 shared more common differentially expressed contigs (147) than did dates 3 and 9 (100) or dates 3 and 12 (63). The contigs shared between dates 9 and 12 included genes involved in cell cycle control, protein metabolism and secondary cell wall deposits: cyclin-dependent protein kinase [36], arginine decarboxylase, and pectinesterase [37] for example. Genes shared between the initial and anabolic stages included more genes related to reactive oxygen metabolism, calcium transport and nitrate transport.

Thirty-three contigs were always differentially expressed on all three dates: five consistently up-regulated and 28 consistently down-regulated (Supplementary file 3 http://tinyurl.com/kkthnev,

Table 3). One consistently up-regulated gene, Cytochrome p450 714a1, has been reported to influence plant development through gibberellin (GA) deactivation [38]. The involvement of GA in nematode parasitism has also been reported in rice (Oryza sativa) where exogenous GA application inhibits gall formation by the root-knot nematode [39]. Up-regulation of Cytochrome p450 714a1 in infected cotton roots may lower GA levels that would otherwise be inhibitory to parasite establishment. Additional members of the cytochrome p450 gene family have also been reported to function in plant defense, such as CYP81D11 [40].

The consistently up-regulated gene glutathione s-transferase (GST) enzymatically conjugates glutathione (GSH) to facilitate reactive oxygen species (ROS) detoxification [41]. Increased ROS detoxification may dampen the ROS signaling that typically characterizes early plant pathogen response [42]. The consistently up-regulated gene transparent testa 12 (TT12) encodes an auxin transporter in Arabidopsis [43]. Flavonoids are ubiquitous plant secondary metabolites with well-characterized roles in both defense and root nodulation, and altering flavonoid distribution by up-regulation of TT12 may promote syncytium formation. Previous work with the gall-forming nematode (Medicago truncatula) indicated that flavonoid-induced galls, underscoring the potential requirement for flavonoids in successful nematode parasitism [44].

Down-regulated contigs included multiple genes related to cell wall synthesis

(beta-d-xylosidase, fasciclin arabinogalactan protein, germin protein, metacaspase, pistil-specific extension, and polygalacturonase), as well as plant defense and stress response genes (germin protein, lrr receptor proteins, non-specific lipid-transfer protein, PIP protein, serine-threonine protein kinases, and subtilisin protease) and genes involved in cellular signaling (phytocyanin,

NAC domain-containing proteins and LRR receptor proteins). Suppression of multiple signaling and stress response genes is likely required to prevent the plant from mounting a successful defense response against the invading nematode, while down-regulation of genes associated with cell wall synthesis may facilitate cell wall lysis and cell-cell interconnection.

Specific gene categories

Cell wall genes – From a single initial endodermis cell, the syncytium expands to include surrounding cells via cell wall degradation and cytoplasmic coalescence [16, 17, 35]. The nematode produces a range of cell wall degrading enzymes to promote this process [45-50]. A number of plant cell wall genes were also down-regulated in our experiment. Extensin, which

13 functions in cell wall assembly and growth [51], was always down-regulated with log2fold change greater than 2. Beta-d-xylosidase, also involved in cell wall modification [50], was down-regulated by at least 1.5 fold on all dates. Other cell-wall related genes, such as pectinesterase, facilitating plant cell wall modification and subsequent breakdown, was up-regualted 0.8 fold on date 3 and 0.5 fold on dates 9 and 12 [37, 52].

In previous work on the syncytium-formation cyst nematode (Heterodera schachtii and H. glycines), expansin, cellulose synthase, and pectin lyase genes were up-regulated in infected roots [22, 46, 53, 54]. Interestingly, these three genes were consistently down-regulated by reniform nematode in our study, suggesting that the two nematode species may induce syncytium formation by fundamentally different molecular processes.

Membrane system modification – A number of genes coding for membrane proteins and transporters were differentially expressed in syncytial roots. A GPI-anchored protein with high sequence similarity to a plasma-membrane-localized lipid transfer protein from Arabidopsis was down-regulated on all three dates [55].

ABC transporter b family has been described as auxin and organic solvent transporters in

Arabidopsis [56, 57]. In previous studies, the enhanced auxin response was visualized at cyst and root-knot nematode feeding sites [58-60] and reduced auxin transport inhibited the radial expansion of syncytium cells with small malformed cyst produced [59, 61]. In our results, ABC transporter was up-regulated with log2fold change ranging from 1.8 to 3.0 across all dates which indicated that the enhanced auxin transporter promoted syncytium formation by reprograming the neighboring cells.

Many various metabolite transporters were significantly up-regulated: sugar transporter, hexose transporter for example [62, 63], was up-regulated with 1.4 log2fold change. Four contigs

14 were annotated as amino acid transporters, two of which were up-regulated with 0.9 log2fold change and the other two with 2.0 log2fold change. Previous studies suggested that amino acid transporters are essential for the H. schachtii and M. incognita and their feeding site development [64, 65].

Changes in membrane-associated proteins and membrane transporters may enhance communication between nearby cells in the syncytium network and promote nutrient transfer to the feeding nematode.

Cell cycle regulation – Reniform syncytium establishment involves a progressive increase in nuclear and cellular size (Figure 3), a phenomenon also observed in the giant cells induced by root-knot nematodes [66]. Unlike multinucleate root-knot giant cells that result from mitosis without cytokinesis [66-68], syncytium cells achieve the network by dissolving the cell walls, so it is possible that less cell cycle control genes are involved in the formation of syncytium cells than giant cells [69, 70]. In our DE analysis, all the cell cycle contigs have been down regulated especially in the late stages when the enlarged cell nuclear showed up and Andreas Niebel et al.

[69] proposed that cyst nematode induce cycles of DNA endoreduplication shunting the M phase.

Our data showed suppressed regulation of G2/M transition of mitotic cell cycle before which the nuclear membrane increases its surface area [71] and this can be observed in the resin section results (Figure 2).

Suppression of defense responses – The nematode must suppress or circumvent host plant defense responses in order to maintain its permanent feeding site [72-74]. Our sequence data showed significant changes in expression of contigs related to pathogen defense responses: 30 contigs were annotated with the “bacterium defense” GO term and 31 with the “fungus defense” GO term. Many plant chemical defenses function against multiple pathogens, and it is

15 possible that the nematode suppresses a suite of plant defenses to ensure its successful developments. Thirty-five differentially expressed genes were annotated as related to the jasmonic acid signaling pathway, and turning off this major defense hormone has been proposed as a possible mechanism for prolonged nematode infection in other systems [74]. A number of genes coding for germin proteins were also down-regulated, and previous researchers have documented the involvement of this protein subfamily in plant defense against fungal and viral pathogens [75]. Germin genes were differentially expressed in in syncytial cells induced by cyst nematodes [76], and Zhang et al [77] proposed germin-like proteins as possible key comonents in the Hs1prol-mediated nematode resistance response.

We also found differential expression of multiple pathogesis-related (PR) proteins in the early stages of syncytium formation. PR proteins have been shown to trigger the accumulation of the hormone salicylic acid (SA), and 31 SA-related genes were also differentially expressed in our study [74, 78]. SA-related genes appeared to be primarily up-regulated at 3 DAI, then down-regulated at 9 DAI and 12 DAI. This pattern is consistent with an early SA-induced signal of pathogen attack, which was subsequently suppressed in later stages of infection to ensure the completion of syncytium development.

Antioxidant enzyme – Reactive oxygen species (ROS) levels often increase dramatically in response to pathogen attack and are thought to serve a signaling function in the plant [79-82].

Melillo et al. [83] found that ROS production increased 12 h after infection in both compatible and incompatible tomato-nematode interactions. In our study, a total of 188 DE contigs were annotated with the GO term “oxidoreductase activity”, most of which were up-regulated as a response to the pathogen or environmental stress. With a nematode defense function, 15 DE contigs were identified as peroxisome in our data and may function in pathogen and elicitor

16 recognition associated with nitric oxide [83].

Metabolic activity – The nematode co-opts significant plant resources for its own development and reproduction, making syncytia large nutrient and carbohydrate sinks [73, 74].

Increased metabolic activity in syncytial cells supports the production of carbohydrates and proteins for the developing nematodes. Up-regulation of 22 ribosomal-associated genes (Cellular component GO-term “ribosome”) on dates 3 and 9 is likely an indicator of the increased translation necessary to maintain rates of metabolism and biosynthesis in syncytial cells.

As the demand for energy and carbon increased with nematode establishment, numerous contigs involved in sugar transportation and translocation were differentially expressed: sugar transporters, hexose transporters, mannitol dehydrogenase, and glucosyltransferase, the latter havng been reported in other parasitic nematode systems [84]. Many genes involved in amino acid metabolism (sulfite reductase, glutamine amidotransferase ylr126c, sphingosine-1-phosphate lyase, phosphatidyl inositol monophosphate 5 kinase, 24-sterol c-methyltransferase, peroxidase 47 and glutamate dehydrogenase 2) and amino acid transport

(ABC transporter family proteins, ammonium transporter, and lysine histidine transporter 1) were strongly-upregulated. The generally high metabolic activity of syncytium was reflected in the large number of differentially expressed contigs annotated with the GO terms “biosynthetic process”, “cellular biosynthetic process”, “cellular metabolic process”, and “macromolecule biosynthetic process” (Figure 7).

Transcription factors – One nac domain-containing proteins (NAC), a homolog of the myb transcription factor ODORANT1, and a c2h2 zinc finger protein (C2H2) were differentially expressed only at 3 DAI, suggesting that they are primarily involved in the early events of reniform infection. Less-studied than the WRKY and ERF transcription factors [85], NAC

17 transcription factors were also the second largest group of differentially-expressed transcription factors. Recently studies have found that viral secreted proteins can interfere with the function of NAC transcription factors [86-88] and suppress the plant defense mechanisms [89]. NAC transcription factors were also differentially expressed during root knot nematode infection of soybean roots [90]. A C2H2-type zinc finger transcription factor is also involved in nodule development on alfalfa roots [91] and in cold tolerance and abiotic stress of soybean [92].

Ethylene-responsive element binding factor (ERF) transcription factors were differentially expressed in syncytial roots and may point to the involvement of the hormone ethylene in syncytium formation. ERFs are known to mediate cross-talk between biotic and abiotic stress-signaling pathways [85, 93]. In Arabidopsis thaliana, ERF transcription factors appear to function synergistically with other transcription factors, including those from the bZIP subfamily, to turn on plant defense-related genes [94]. bZIP transcription factors were also differentially-expressed in our work. Muhammad et al. reported that overexpression of the ERF transcription factor RAP2 enhanced callose deposition in syncytia and increased to beet cyst nematode infection [95].

WRKY transcription factors are involved in multiple aspects of plant biology, including senescence, abiotic and biotic stress response, pathogen defense and plant immunity [85, 96,

97]. In Arabidopsis, both up- and down- regulation of specific WRKY transcription factors were reported in cyst nematode syncytia. Most were significantly down-regulated, and the authors suggested that their down-regulation interfered with plant defense reactions [98].

The largest number of differentially expressed transcription factors in our experiment belonged to the zinc finger family, members of which have been also been implicated in other nematode-plant systems [90, 99]. Zinc finger proteins are a very large family with multiple roles

18 in the plant [100]. Future work with virus-induced gene silencing may shed light on the specific functions of these differentially-expressed transcription factors in syncytium formation.

In expression level analysis, transcription factor bhlh (bHLH), transcription factor lux (LUX), wrky transcription factor (WRKY), and myb domain protein (MYB) were differentially expressed with log2fold of 2 or greater. Besides, zinc finger family is the largest DE transcription family taking all the different types into consideration. And previous nematode studies also reported differential expressions [90, 99]. With a vast majority typically functions as interaction modules that bind DNA, RNA and proteins, zinc finger protein may involve in many transcriptional regulation of different genes [100].

In conclusion, we have now identified over 1000 cotton genes whose expression is significantly up- or down-regulated during the process of reniform syncytium formation. Some of these genes have previously been reported in the development of cyst nematode syncytia and root knot nematode giant cells, suggesting that common genetic mechanisms may underlie feeding site induction by phylogenetically-distinct nematodes. In future work, we will use gene network analysis to narrow down this candidate gene list and select a subset of genes for functional characterization with RNAi, virus induced gene silencing, and fluorescent staining of target proteins. We thereby hope to identify genes whose manipulation through traditional breeding or genetic engineering will result in reniform-nematode resistant cotton plants.

Table 1 Summary of sequencing and assembly

Sequencing Inoculated Non-inoculated Total Average Raw Reads 224,016,632 224,557,062 448,573,694 --- Clean Reads 193,057,752 220,976,242 414,033,994 --- N percent 0.0% 0.0% --- 0.0% GC percent 50.3% 49.6% --- 50.0%

Assembly Total number of assembled contigs 213,984 Number of unique contigs 156,156 Number of alternate splice forms 57,828 Mean Contig Length (± SD) 546 (± 602) Contig N50 712

Table 2. Top twenty up- and down-regulated genes on each sampling date. Sequence descriptions are based on top BLASTx hits for each contig. Normalized counts are expressed as number of reads mapping to the contig per million mapped reads (N=3 for each date x infection status combination). * means the same or similar contigs show multiple times in same subtable.

A. Three days after inoculation Top twenty up-regulated contigs

Contig Name Sequence Description1 Normalized counts Normalized counts Notes Control Syncytial comp134769 Cytochrome p450 protein 1 ± 1 105 ± 80 comp132194 Protein srg1 0 90 ± 97 comp56797 Desiccation-related protein pcc13-62 0 19 ± 4 Also top up-regulated date 9 comp145526 Mlo protein homolog 1 1 ± 1 34 ± 8 * comp136200 Mlo protein homolog 1 2 ± 3 56 ± 16 * comp115774 Short vegetative phase protein 0 11 ± 3 comp92021 Protein nrt1 ptr family 0 33 ± 32 comp116534 Hgh-affinity nitrate transporter 52 ± 63 1374 ± 469 Also top up-regulated date 9 and 12 top down-regulated date 12 comp106962 Short chain alcohol 9 ± 11 185 ± 70 comp130253 Elongation factor 3 0 24 ± 27 Also top up-regulated date 9 comp102937 ABC transporter b family member 15 36 ± 36 491 ± 169 Also top up-regulated date 12 comp106738 Suppressor of overexpression of co1 like protein 3 ± 0 34 ± 11 comp120559 Type i inositol trisphosphate 5-phosphatase cvp2 3 ± 1 43 ± 26 comp104742 Ethylene-responsive transcription factor 1 ± 0 14 ± 6 * comp71229 AP2 ethylene-responsive transcription factor at1g16060 0 ± 1 11 ± 9 * comp110481 Ammonium transporter 0 10 ± 5 Also top up-regulated date 9 comp93575 Quinone oxidoreductase pig3 1 ± 1 15 ± 3 comp107637 Wall-associated receptor kinase 20 1 ± 1 28 ± 25 comp89247 MYB domain protein 1 ± 1 18 ± 15 comp133561 High-affinity nitrate transporter 2 ± 2 22 ± 1 Also top up-regulated date 9 and 12 top down-regulated date 12

Top twenty down-regulated contigs

Contig Name Sequence Description1 Normalized counts Normalized counts Notes Control Syncytial comp127470 Beta-d-xylosidase 5 84 ± 10 7 ± 6 comp102141 Alpha-expansin 6 18 ± 4 1 ± 2 comp104500 Phosphate-responsive 1 family protein 43 ± 2 4 ± 3 comp122058 Protein hothead 35 ± 14 3 ± 1 comp105034 Mannan endo-beta-mannosidase 6 mRNA 35 ± 2 3 ± 2 comp88048 Limonoid udp-glucosyltransferase 34881 ± 14234 3952 ± 870 comp104850 Pistil-specific extensin 16 ± 1 2 ± 1 Also top down-regulated date 9 and 12 comp112747 Laccase family protein 18 ± 3 2 ± 2 Also top down-regulated date 9 comp89519 Low quality protein: uncharacterized loc101220997 54 ± 60 2 ± 2 comp64631 Non-specific lipid-transfer protein 2 23 ± 5 2 ± 2 Also top down-regulated date 9 and 12 comp117308 ATP-dependent RNA helicase ddx11 665 ± 47 92 ± 33 comp151125 Pectate lyase 20 ± 6 2 ± 1 Also top down-regulated date 12 comp39701 Late embryogenesis abundant protein d-34 23 ± 11 1 ± 1 comp104162 Heavy metal-associated isoprenylated plant protein 26 170 ± 84 23 ± 10 comp109158 Alcohol dehydrogenase 1 32 ± 21 1 ± 2 Also top up-regulated date 9 comp111788 Germin protein subfamily 3 member 4 75 ± 5 7 ± 7 Also top down-regulated date 9 and 12 comp130136 DUF579 protein 37 ± 12 4 ± 2 comp124712 Cellulose synthase 30 ± 0 4 ± 2 * comp125725 Cellulose synthase a catalytic subunit 7 53 ± 28 6 ± 2 * comp113469 Peroxidase 66 390 ± 42 51 ± 32 Also top up-regulated date 12

B. Nine days after inoculation Top twenty up-regulated contigs

Contig Name Sequence Description1 Normalized counts Normalized counts Notes Control Syncytial comp64922 Conserved hypothetical protein 76 ± 43 1371 ± 282 comp101761 tRNA--methyltransferase non-catalytic 8 ± 4 102 ± 6

Subunit trm6MTase subunit comp122571 Mitochondrial phosphate carrier protein 2 ± 3 42 ± 14 comp129671 Glutamate receptor 25 ± 2 212 ± 68 comp64524 Conidiation-specific protein 6 219 ± 123 1995 ± 160 comp63827 Niban protein 1 1 ± 1 46 ± 50 comp110481 Ammonium transporter 15 ± 12 195 ± 135 Also top up-regulated date 3 comp118531 Anb1p 3 ± 5 80 ± 60 comp99476 Alcohol dehydrogenase 11 ± 3 81 ± 0 Also top down-regulated date 3 comp126913 Translation elongation factor 1-beta 5 ± 5 70 ± 37 Also top up-regulated date 3 comp121989 F-box protein at5g07610 8 ± 3 64 ± 16 comp150815 UDP-glycosyltransferase superfamily 16 ± 6 108 ± 9 comp118886 Actin partial 10 ± 13 203 ± 210 comp97008 Basic transcription factor 3 isoform 1 1 ± 2 47 ± 51 comp84452 Sulfite reductase 121 ± 17 911 ± 166 comp92812 Flowering-promoting factor 1 protein 3 32 ± 17 228 ± 50 Also top up-regulated date 12 comp121190 Short-chain dehydrogenase reductase 2a 10 ± 4 83 ± 65 comp115409 Pentatricopeptide repeat superfamily isoform 1 20 ± 2 397 ± 544 comp56797 Desiccation-related protein pcc13-62 12 ± 11 333 ± 443 Also top up-regulated date 3 comp114135 Protein ELC 20 ± 6 111 ± 17 Also top up-regulated date 12

Top twenty down-regulated contigs

Contig Name Sequence Description1 Normalized counts Normalized counts Notes Control Syncytial comp100257 At2g10940 protein 303 ± 161 14 ± 8 comp104850 Pistil-specific extension 352 ± 103 23 ± 19 *Also top down-regulated date 3 and 12 comp127614 Leucine-rich repeat extensin protein 2 485 ± 254 39 ± 7 *Also top down-regulated date 3 and 12 comp108018 Lipid transfer protein vas 46 ± 34 1 ± 1 Also top down-regulated date 3 and 12 comp131902 Beta-amylase 1 180 ± 20 19 ± 11 comp97650 Germin protein subfamily 3 member 2 73 ± 46 4 ± 4 Also top down-regulated date 3 and 12 comp90434 Fiber protein fb10 38 ± 21 2 ± 1

23 comp116548 Osmotin protein 2063 ± 783 234 ± 104 Also top down-regulated date 12 comp120518 Pectinesterase pectinesterase inhibitor 40 92 ± 90 4 ± 4 Also top down-regulated date 12 comp89427 Glucuronoxylan 4-o-methyltransferase 1 47 ± 28 3 ± 4 comp111791 Lysine histidine transporter 8 86 ± 32 9 ± 5 Also top down-regulated date 12 comp103343 Lipid-transfer protein dir1 2046 ± 973 256 ± 14 *Also top down-regulated date 3 and 12 comp106603 Laccase 2 34 ± 30 1 ± 1 Also top down-regulated date 3 comp64631 Non-specific lipid-transfer protein 2 1324 ± 552 155 ± 80 *Also top down-regulated date 3 and 12 comp94877 Homeobox protein knotted-1 7 18 ± 2 1 ± 1 comp116591 Auxin efflux carrier component 6 237 ± 78 29 ± 10 Also top down-regulated date 12 comp127510 NAC domain-containing protein 7 53 ± 10 8 ± 3 comp111421 RAB6-interacting golgin 121 ± 63 15 ± 2 comp108534 IQ motif and sec7 domain-containing protein 2 43 ± 15 4 ± 1 comp103364 Pistil-specific extensin 1988 ± 434 326 ± 31 *Also top down-regulated date 3 and 12

C. Twelve days after inoculation Top twenty up-regulated contigs

Contig Name Sequence Description1 Normalized counts Normalized counts Notes Control Syncytial comp38703 Peroxygenase 2 isoform 1 6 ± 2 48 ± 32 comp129458 Phosphoenolpyruvate carboxylase 4 ± 1 36 ± 21 comp127724 Glutathione s-transderase ul complete cds 195 ± 97 1076 ± 481 comp104553 Flowering-promoting factor 1 protein 3 28 ± 25 204 ± 78 Also top up-regulated date 9 comp102740 Hypersensitive-induced response protein 6 ± 3 37 ± 15 comp101859 Uncharacterized protein TCM_033176 36 ± 10 163 ± 59 * comp110390 Vacuolar iron transporter homolog 4 14 ± 6 72 ± 40 comp102937 ABC transporter b family member 15 478 ± 179 1993 ± 515 Also top up-regulated date 3 comp63492 Phytosulfokines 3 36 ± 39 227 ± 56 comp115118 Nodulation-signaling pathway 2 11 ± 5 46 ± 4 comp114135 Protein ELC 34 ± 7 131 ± 31 Also top up-regulated date 9 comp132597 Endochitinase pr4 136 ± 60 562 ± 184 comp123585 FAD-dependent urate hydroxylase 136 ± 85 579 ± 43

24 comp127117 Aluminum-activated malate transporter 10 10 ± 5 245 ± 94 comp91931 Uncharacterized protein TCM_045917 10 ± 5 87 ± 109 * comp122422 ATP synthase subunit alpha 710 ± 280 4392 ± 3791 comp99919 Stress induced protein 68 ± 16 272 ± 141 comp114316 Peroxidase 10 159 ± 136 905 ± 255 Also top down-regulated date 3 and 12 comp105250 ELF4 protein 13 ± 4 55 ± 20 comp38904 Nitrate transporter 9 ± 2 36 ± 2 Also top up-regulated date 3 top down-regulated date 12

Top twenty down-regulated contigs

Contig Name Sequence Description1 Normalized counts Normalized counts Notes Control Syncytial comp104850 Pistil-specific extensin 421 ± 156 37 ± 16 Also top down-regulated date 3 and 12 comp98012 Protein radialis 1 101 ± 79 4 ± 1 comp120518 Pectinesterase pectinesterase inhibitor 40 71 ± 39 6 ± 3 Also top down-regulated date 9 comp121369 Peroxidase 52 87 ± 63 4 ± 5 *Also top down-regulated date 3 top up-regulated date 12 comp64631 Non-specific lipid-transfer protein 2 1117 ± 378 203 ± 94 *Also top down-regulated date 3 and 9 comp127614 Leucine-rich repeat extensin protein 2 488 ± 367 57 ± 38 *Also top down-regulated date 3 and 9 comp151125 Pectate lyase 139 ± 119 10 ± 9 Also top down-regulated date 3 comp94679 Non-specific lipid-transfer protein 2 2961 ± 998 618 ± 210 *Also top down-regulated date 3 and 9 comp97650 Germin protein subfamily 3 member 2 134 ± 68 24 ± 9 Also top down-regulated date 3 and 9 comp116591 Auxin efflux carrier component 6 205 ± 58 44 ± 14 Also top down-regulated date 9 comp114852 Nitrate transporter 42 ± 4 8 ± 5 Also top up-regulated date 3 and 12 comp111791 Lysine histidine transporter 8 93 ± 48 17 ± 6 Also top down-regulated date 9 comp113485 Non-specific lipid-transfer protein 1745 ± 827 370 ± 94 *Also top down-regulated date 3 and 9

25 comp124777 Zinc finger protein nutcracker 233 ± 113 47 ± 17 comp135702 Bacterial-induced peroxidase 133 ± 90 23 ± 9 *Also top down-regulated date 3 top up-regulated date 12 comp101941 14 kDa proline-rich protein 219 ± 37 38 ± 29 comp116548 Osmotin protein 2373 ± 795 606 ± 111 Also top down-regulated date 9 comp83891 kDa class i heat shock family protein 167 ± 61 40 ± 9 comp125205 WEB family protein at2g40480 41 ± 11 9 ± 4 comp112711 Plasma membrane intrinsic protein 175 ± 30 45 ± 13

Table 3: Thirty-three genes differentially expressed in syncytial roots across all sampling dates.

Contig ID Sequence Description Normalized counts (control) Normalized counts (syncytial) Notes 3 DAI 9 DAI 12 DAI 3 DAI 9 DAI 12 DAI Up-regulated comp102937 ABC transporter b family member 15 36±36 1073±410 478±179 491±169 4445±1925 1993±515 Abiotic stress, auxin transport [41, 56, 57, 108] comp93536 BEL1 homeodomain protein 2 13±6 113±20 82±30 46±17 439±95 244±112 Transcription factor [109-111] comp120864 Cytochrome p450 714a1 18±14 103±39 68±21 72±29 376±56 154±44 Plant defense, gibberellin deactivation [38, 40] comp127724 Glutathione s-transferase ul complete cds37±32 268±91 195±97 367±233 1463±1038 1076±481 ROS defense [41, 79, 112] comp114455 Protein transparent testa 12 80±21 150±51 150±41 206±60 615±201 395±57 Flavonoid sequestration [43, 113, 114]

Down-regulated comp127470 Beta-d-xylosidase 5 390±42 295±348 364±202 51±32 20±14 89±40 Cell wall structure [115, 116] comp118969 Fasciclin arabinogalactan 44±14 55±19 66±27 8±4 7±3 19±6 Secondary cell wall protein 7 isoform 1 [117-119] comp90434 Fiber protein fb10 18±7 38±21 40±31 3±1 2±1 4±3 Human Hsp27 homolog [120] comp97650 Germin protein subfamily 3 member 2 84±3 73±46 134±68 15±12 4±4 24±9 Defense, cell wall remodeling [75-77, 121-124] comp122032 Inactive purple acid phosphatase 29 179±57 219±65 276±64 62±27 58±29 102±22 Binuclear ion proteins [125] comp111918 LRR receptor protein kinase at5g49770 60±24 73±25 69±35 11±2 10±2 17±4 Disease resistance, comp99342 LRR receptor protein kinase at5g49770 42±10 60±24 55±26 8±2 14±6 15±2 stress response, comp107156 LRR receptor serine threonine-protein 65±9 147±33 144±31 35±5 50±1 61±20 pathogen recognition kinase at3g47570 signaling [112, 126-134] comp111791 Lysine histidine transporter 8 50±5 86±31 93±48 21±5 9±5 17±6 Amino acid uptake [135] comp105034 Mannan endo-beta-mannosidase 6 75±5 62±50 70±43 7±7 6±3 16±5 Hyrolyse O- and S-glycosyl compound [136]

27 comp104961 Mavicyanin 843±117 1322±484 1398±581 308±140 275±96 485±139 Redox chemical [137] comp111314 Metacaspase 9 168±62 147±127 128±81 33±16 20±12 27±18 Cell disassembly; PCD [138, 139] comp127510 NAC domain-containing protein 7 57±15 63±10 63±28 11±3 8±3 21±5 Transcription factor, stress response, and auxin signaling [88, 90] comp113485 Non-specific lipid-transfer protein 1115±5 1685±770 1745±827 262±59 271±250 370±94 Phospholipid transfer, comp64631 Non-specific lipid-transfer protein 2 665±47 1324±552 1117±378 92±33 155±80 203±94 defense and stress comp94679 Non-specific lipid-transfer protein 2 1319±149 2362±1346 2961±998 335±68 307±181 618±210 response, and hydrophobic protective layers [140-143] comp104966 Phytocyanin 86±0 217±45 196±8 41±5 46±3 89±14 Signaling, defense [144, 145] comp80448 PIP protein 191±45 370±155 350±76 66±11 84±21 137±11 Stress response comp95130 PIP protein 193±44 376±142 371±90 65±34 71±4 135±30 [146-148] comp103364 Pistil-specific extensin 1043±515 1988±434 2720±212 368±92 326±31 916±98 Cell wall strengthen, comp104850 Pistil-specific extensin 170±84 352±103 421±156 23±10 23±19 37±16 stress response and defense [51, 149] comp117969 Polygalacturonase non-catalytic 436±174 392±99 403±153 128±48 102±27 164±14 Cell adhesion [150] subunit jp650 comp119484 Serine threonine-protein kinase 171±5 336±221 302±113 76±31 54±15 79±31 Wound-induced, immunity signaling [90] comp125794 Subtilisin protease 280±93 378±248 511±260 65±5 84±50 180±60 Defense [151-154] comp78258 Tetratricopeptide repeat 57±5 75±40 99±58 16±2 17±5 22±6 Act with actin binding superfamily protein domain [155] comp111509 Uncharacterized gpi-anchored 132±23 250±68 278±45 70±11 74±10 93±19 Plasma membrane [55] protein at1g27950 comp108695 Uncharacterized loc101209149 331±84 635±157 572±131 169±17 159±72 218±28 ---- comp125205 WEB family protein at2g40480 22±10 43±18 41±11 3±4 4±3 9±4 -----

Ne SC

3DAI 9DAI

12DAI

Figure 2. Resin section of reniform nematode penetration and syncytium formation. Syncytium cells were circled in red, and non-syncytium cells in yellow. DAI: days after inoculation; Ne: reniform nematode; SC: syncytium cells (circled in red line).

Figure 3. Unique contigs annotation results from BLAST2GO. Contigs with top BLASTed plant species were regarded as putative cotton contigs, contigs with top BLASTed animal species were regarded as putative animal contigs, and other contigs include all top BLAST hits assigned as fungi, bacteria, and virus and so on.

Figure 4. DE contigs distribution on different dates. Pink bars present the significantly up-regulated contigs, and blue bars present the significantly down-regulated contigs.

Figure 5. Distribution of expression changes on different dates. Black dots show non-DE contigs, colored dots show all the DE contigs significant regulated with log2fold of 2 or greater presented as green.

Figure 6. Common DE contigs shared between different dates. Date-specific contig expression profiling with Venn Diagram with each section showing the contig expression number.

Figure 7. GO annotation of DE unique contigs by BLAST2GO. The x-axis indicated the sub-categories and the y-axis indicated the number of unique contigs, and the unique contig number assigned with same GO terms were indicated on the top of each group (GO terms with more than 5 presented here). A for biological process, B for cellular component, and C for molecular function.

Figure 8. DE transcript factor

APPENDICES

Appendix A

Supplementary Figure 1

Supplementary Figure 1. PCA plot. X3.N.1 and X3.N.2 presented noninoculated root tissue data for 3DAI, X3.IN.1, X3.IN.2 and X3.IN.3 presented inoculated root tissue data for 3DAI. X9.N.1,

X9.N.2 and X9.N.3 presented noninoculated root tissue data for 9DAI, X9.IN.2 and X9.IN.3 presented inoculated root tissue data for 9DAI. X12.N.1, X12.N.2 and X12.N.3 presented noninoculated root tissue data for 12DAI, X12.IN.1, X12.IN.2 and X12.IN.3 presented inoculated root tissue data for 12DAI. Since only few reads were sequenced for X3.N.3 and X9.IN.1, those two data groups have been discarded.

Appendix B

Supplementary File 1: Full Transcriptome Annotation

(http://tinyurl.com/kkjc5b3)

Appendix C

Supplementary File 2: Normalized mapped read counts for protein coding cotton genes

(http://tinyurl.com/llutj3v)

Appendix D

Supplementary File 3: 33 common contigs Annotation

(http://tinyurl.com/kkthnev)

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Reniform Nematode (&lt;I&gt;Rotylenchulus

Reniform Nematode (<I>Rotylenchulus