Accepted Manuscript Rice-gall midge interactions: battle for survival Jagadish S. Bentur, Nidhi Rawat, D. Divya, Deepak K. Sinha, Ruchi Agarrwal, Isha Atray, Suresh Nair PII: DOI: Reference:

S0022-1910(15)00198-5 http://dx.doi.org/10.1016/j.jinsphys.2015.09.008 IP 3438

To appear in:

Journal of Insect Physiology

Received Date: Revised Date: Accepted Date:

17 December 2014 31 July 2015 14 September 2015

Please cite this article as: Bentur, J.S., Rawat, N., Divya, D., Sinha, D.K., Agarrwal, R., Atray, I., Nair, S., Ricegall midge interactions: battle for survival, Journal of Insect Physiology (2015), doi: http://dx.doi.org/10.1016/ j.jinsphys.2015.09.008

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Rice-gall midge interactions: battle for survival

Jagadish S. Bentur2†, Nidhi Rawat2¶, D. Divya 2, Deepak K. Sinha1, Ruchi Agarrwal1, Isha Atray1, Suresh Nair1*

1

International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg,

New Delhi 110 067, India. 2

Directorate of Rice Research, Rajendranagar, Hyderabad 500 030, India

Current address: †

Agri Biotech Foundation, Rajendranagar, Hyderabad 500 030, India

¶

Gulf Coast Research & Education Center, University of Florida, FL 33598, USA

*

Corresponding author: Suresh Nair, International Centre for Genetic Engineering and

Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 067, India. Email: [email protected] Telephone: 91-11-26741242; Fax: 91-11-26742316

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ABSTRACT Gall midges are insects specialized in maneuvering plant growth, metabolic and defense pathways for their benefit. The Asian rice gall midge and rice share such an intimate relationship that there is a constant battle for survival by either partner. Diverse responses by the rice host against the midge include necrotic hypersensitive resistance reaction, nonhypersensitive resistance reaction and gall-forming compatible interaction. Genetic studies have revealed that major R (resistance) genes confer resistance to gall midge in rice. Eleven gall midge R genes have been characterized so far in different rice varieties in India. In addition, no single R gene confers resistance against all the seven biotypes of the Asian rice gall midge, and none of the biotypes is virulent against all the resistance genes. Further, the interaction of the plant resistance gene with the insect avirulence gene is on a gene-for-gene basis. Our recent investigations involving suppressive subtraction hybridization cDNA libraries, microarray analyses, gene expression assays and metabolic profiling have revealed several molecular mechanisms, metabolite markers and pathways that are induced, downregulated or altered in the rice host during incompatible or compatible interactions with the pest. This is also true for some of the pathways studied in the gall midge. Next generation sequencing technology, gene expression studies and conventional screening of gall midge cDNA libraries highlighted molecular approaches adopted by the insect to feed, survive and reproduce. This constant struggle by the midge to overcome the host defenses and the host to resist the pest has provided us with an opportunity to observe this battle for survival at the molecular level.

Key words: Rice gall midge; Orseolia oryzae; Diptera; Cecidomyiidae; R genes; Avr genes; Insect-plant interaction; Biomarkers; Metabolomics; Metabolic profiling; Rice-gall midge interaction; Expressed sequence tags (ESTs); Gene expression; Secreted salivary gland proteins (SSGPs); Serine proteases; Next generation sequencing (NGS); Transcriptomics;

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Introduction Evolution of plant and phytophagous arthropods is directed towards their ability to either avoid or suppress or reprogram host metabolic machinery to favor their survival. Essentially, this "arms race" is an ongoing phenomenon and the ability to rapidly respond and evolve is the key to success of both the interacting partners (i.e. host plant and herbivorous insect). Plants are endowed with a large array of defense mechanisms that are either basal in nature or induced upon herbivore attack. Both these types of mechanisms are highly regulated and controlled by various regulators or regulatory events. Similarly, the phytophagous insects have evolved different ways to defend against both the types of host defenses and subsequently hijack the plant metabolism by creating a suitable environment for their continued feeding and growth.

Among the phytophagous insects, gall formers or gall midges are exemplars in evading or maneuvering the plant defense metabolism. They divert host nutrients for their feeding and orient hormone signaling to reconfigure the host cell morphology leading to the formation of galls (abnormal outgrowth of plant tissues). In food crops such as wheat and rice, formation of these galls renders the tiller sterile thereby causing huge economic loss. Hessian fly (Mayetiola destructor Say) and the Asian rice gall midge (AGM; Orseolia oryzae WoodMason) are two such well-studied gall midges infesting wheat and rice, respectively. Both the Hessian fly and AGM belong to Diptera: Cecidomyiidae but the former belongs to supertribe Lasiopteridi while the latter to Cecidomyiidae. The Hessian fly larva feeds at the crown or nodes of the host plant and does not induce formation of any macroscopic gall-like structure but since it forms a nutritive tissue at the feeding site (Harris et al. 2006), therefore it is classified as a gall midge. The Hessian fly is believed to have originated from southwest Asia but is a major pest of wheat in United States and North America as well. On the other hand AGM is a serious pest of rice in south and south-east Asia while the African gall midge O. oryzivora (Harris and Gagne) is predominant in Africa.

In India, AGM is rated the third most important pest of rice following stem borers and planthoppers. Among these, AGM is the only one that is capable of extensively manipulating its host for its own survival. The latter two groups represent complex of species. There are at

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least five species of stem borers representing families Pyralidae and Noctuidae. The yellow stem borer (YSB), Scirpophaga incertulas (Walker) is the most widespread and devastating. Host range of YSB is limited to Oryza genus, though cultivated rice is most preferred. Direct damage to plant tissue is inflicted by insect feeding and there is no recovery of the damaged tissue. Plant defense against this pest is not widely reported, though some wild relative species have been reported to show limited damage (Khan et al. 1991). The only response of plant appears to be a circular necrotic ring at the base of panicle in the first internode that may reflect a hypersensitive reaction (HR) of a sort. Earlier studies reported that high level of silica in rice varieties such as TKM6 causes wear and tear of larval mandible and thereby, provides mechanical protection against the insect feeding (Heinrichs, 1994). No plant resistance genes against YSB have been reported so far.

The planthhoppers are represented by three delphacids: brown planthopper (BPH), Nilaparvata lugens (Stål); whitebacked planthopper (WBPH), Sogatella furcifers (Horvath) and smaller brown planthopper (SBPH) Laodelphax striatellus (Fallén). Often, when population crosses a threshold density, it explodes beyond the carrying capacity of the habitat. Such events lead to ‘hopper burn’ causing total loss of the crop. Planthoppers cause both direct damage to the plant by sucking the phloem sap and indirect damage through transmission of viruses. However, there is no manipulation of the host as such. Genetic resistance against all the three species has been reported through large scale screening of rice germplasm under greenhouse conditions (Heinrichs et al. 1985, Bentur et al. 2011). Such a screening method has mainly identified genotypes that are less preferred and are less fed upon (Horgan, 2009). However, certain genotypes exhibit ovicidal activity i.e. by killing the eggs laid by WBPH. Few of the BPH resistant rice genotypes exhibit tolerance mechanism by enhancing photosynthesis to compensate loss inflicted by the pest. So far 29 BPH resistance genes and several QTLs have been identified from different germplasm accessions of cultivated indica subspecies of O. sativa and from the wild relatives such as O. australiensis, O. officinalis, O.minuta, O. eichengeri, O. rufipogan, O. latifolia and African cultivated O. glaberrima (Cheng et al. 2013, Fujita et al. 2013, Wang et al. 2015). These studies have shown that though resistance to planthoppers is wide spread and distributed widely among Oryza species, resistance is quantitative.

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A vast majority of the cultivated high yielding rice varieties is prone to AGM attack, but many of the cultivars and land races are immune to it. On resistant rice varieties maggots make their way to the apical meristem, but none survive beyond 2-4 days after hatching and fail to induce gall formation. Though such varietal resistance was reported over a century ago, modern rice breeding for gall midge resistance was initiated only during late 1950s and the first gall midge resistant rice variety developed through hybridization was released for cultivation in 1975. Field evaluation of local rice germ plasm in these two decades identified several sources of resistance such as Eswarakora, Siam29, Ptb10, Ptb18 and several others, which were used in developing new resistant cultivars (Bentur et al. 2013). Even during these early phases, differential reaction of the same rice genotype against gall midge populations at different rice growing locations was noticed and led to the identification of different gall midge biotypes (GMB), as geographically distinct populations (Khan and Murthy, 1955). While extensive manipulation of the host plant by AGM has been described in earlier literature, systematic studies on the genetic basis of resistance were initiated during 1970s and molecular basis of resistance and susceptibility only during last two decades.

Biology of the Asian rice gall midge and the damage caused to host Like a typical cecidomyiid, AGM has three instars as maggots prior to pupation. Eggs are laid on plant surface without discretion as host plant and hatch on 4th day after oviposition under humid environment. Neonate maggots crawl in the thin film of water on the surface of leaf sheaths to enter the space between these. This stage is critical and success depends on humidity and rainfall. First two instars feed actively while feeding ceases in the third instar. Larval duration lasts about 8-10 days while pupal duration is about 10-12 days depending on the ambient temperature (Sain, 1988). AGM pupa is exceptionally active and wriggles upwards in the elongated gall cavity and drills an exit hole at the apex and slightly protrudes out to facilitate eclosion and adult emergence. First instar maggots lack fat bodies when they hatch and with the help of sternal spatula (Supplementary Fig. 1) begin feeding on the apical meristematic tissues of the host. At the feeding site, the host cells enlarge and show hypertrophy and hyperplasia (Sain, 1988) establishing a nutritive tissue for insect feeding. While several maggots reach the meristem, soon one of them predominates while others are pushed out of the gall chamber that is built around the active maggot. Rest of the maggots survive without feeding for some time and move to the new tillers that are actively produced by the plant. The gall chamber insulates the maggots and later elongates as a tubular structure

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when feeding by larvae stops. Gall is the modified leaf sheath that turns tubular in shape and bears vestigial leaf blade at the top. This change arrests further differentiation of the tillers and formation of reproductive structures leading to yield loss. However, infested plant tries to compensate the damage by producing more tillers that are often attacked by the surviving maggots. AGM is predominantly a pest of wet (kharif) season and in irrigated and rainfed rice. This is due to eggs and maggots being sensitive to humidity, as mentioned above. It is distributed over most of the rice growing countries of south and south-east Asia with few exceptions such as the Philippines and Pakistan. Within a region, however, certain areas have been known to be pest endemic with regular occurrence and concomitant yield loss. Besides weather, prevailing cultivars and intensity of cropping influence the pest cycles. Introduction of resistant rice varieties in such areas followed by emergence of virulent biotypes often resulted in “buck and boost” cycles of pest recurrence. Estimation of yield losses due to AGM has often been tricky and based on “control” vs “protected” crop. Two independent studies during 1990s overcoming these biases estimated the loss due to AGM to be about 0.8% of total yield or approximately US$ 80.00 million in South India (Bentur et al. 2003).

The Gene-for-Gene concept and gall midge resistance in rice Through intensive studies on genetics of resistance spanning from Chaudhary et al. (1985) to Himabindu et al. (2010), it emerged that resistance in rice against the gall midge is simply inherited and conferred by major R (resistance) genes, mostly dominant in nature. The defense in the plant is triggered by the interaction between products of the resistance (R) gene from the plant and the avirulence (avr) gene from the insect. One of the most effective methods of curbing the infestation is through the development of resistant rice cultivars. However, extensive cultivation of these resistant varieties has led to the evolution of new virulent biotypes, further aggravating the problem. Also, studies on inheritance of virulence in biotypes suggested a single recessive gene conferring virulence (Bentur et al. 1992). Some of the virulence genes are sex-linked (Behura et al. 2000, Bentur et al. 2008). Thus a genefor-gene relation was evident between rice R genes and gall midge biotypes (Nair et al. 2011), as evident between the Hessian fly biotypes and wheat R genes (Harris et al. 2003). So far 11 R genes in rice (Himabindu et al. 2010) and seven distinct gall midge biotypes (Vijaya Lakshmi et al. 2006) have been characterized in India. While none of the R genes confers

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resistance against all the seven biotypes, none of the biotypes is virulent against all the R genes.

The observation that HR is associated with some, but not all, R genes indicated the diversity in nature of resistance conferred by different R genes (Bentur and Kalode, 1996). Rate of adaptation in pest population against such diverse genes was also noted to differ even in the same population (Bentur et al. 2008, Andow and Bentur, 2010). It is thus evident that evolution of resistance in rice and counter evolution of virulence in gall midge is yet another example of battle for survival. The insect reprograms the host for its survival while plant deploys counter offensive through suppression of insect defense or through the production of feeding-deterrents or toxic compounds.

The following review, mostly covering our recent work on rice-gall midge interactions, highlights alternative pathways of plant defense against the insect and some of the pest’s offensive arsenals that target plant morphogenesis and differentiation. While several of the candidate R genes have been cloned, stage is set for precise characterization of candidate gene products and their specific action and interactions with products of counter acting genes. In this process rice-gall midge system has emerged as yet another model system besides the wheat-Hessian fly system. Data obtained from studies on this interaction, provides an outline of rice defense with reference to gene expression, metabolomics and a perspective from pest side, from the viewpoint that the pest manipulates the host for its survival and continuity.

Gall midge resistance genes in rice Search for new sources of gall midge resistance has been a continuous endeavor through field screening and greenhouse evaluation (Bentur et al. 2013, Nair et al. 2011). Over 35,000 accessions of rice germ plasm covering primary and secondary gene pools have been evaluated to identify over 600 sources of resistance. Through national screening activity several of these accessions have been tested across pest endemic locations. In addition, as a result of intensive studies on genetics of resistance in a small subset of the screened accessions, 11 R genes have been characterized (Himabindu et al. 2010), of which gm3 is the only recessive R gene identified thus far. Based on the spectrum of resistance conferred by R genes across the seven biotypes these could be grouped into four groups (Bentur et al. 2013; Table 1). Several of the resistant genotypes did not fit the criteria for classification and

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therefore did not fall into any of the four groups. It is thus reasonable to assume that many more R genes remain to be characterized. Grouping of several resistance genes such as Gm2, Gm5, Gm6, Gm7 and Gm9 in one group suggested common spectrum of resistance conferred by these genes. However, it is not clear if these genes are duplicates that are lodged in different regions of the rice genome, or whether they possess subtle variations in their mode and spectrum of resistance which needs to be investigated in the future.

We initiated tagging and mapping of the gall midge resistance genes (Mohan et al. 1994, Nair et al. 1995) and till date eight of the 11 R genes (Gm1, Gm2, gm3, Gm4, Gm6, Gm7, Gm8 and Gm11) have been tagged and mapped on to different chromosomes (Nair et al. 2011) of rice. Gm5 has been tagged but not mapped. Three of these genes were further fine mapped based on candidate-gene-based markers (Yasala et al. 2012). Further, candidate genes for gm3 (Sama et al. 2014) and Gm4 (Divya et al. 2015) have been functionally validated. Both of the candidate genes belong to Nucleotide Binding Site-Leucine-Rich Repeat (NBS-LRR) class of R genes widely implicated in plant resistance to pathogens (De Young and Innes, 2006) and insects (Du et al. 2009). Candidate for the third gene, Gm8, is likely to be a proline rich protein that is being functionally validated (Divya et al. unpublished). While map-based approach led to the identification of candidate genes, this did not elaborate the molecular basis and diversity of resistance in rice against gall midge. Therefore, genome-wide gene expression profiling and metabolic profiling was also performed to elucidate the resistance mechanism(s) and unveil the molecular processes that were activated/suppressed/regulated within the rice host in response or due to the gall midge infestation.

Host defense and reprogramming Plant response to insect herbivores is mediated through a suit of genes, which is modulated either in favor of the plant or of the insect (Stuart et al. 2012, War et al. 2012). Dramatic reprogramming of gene expression in plants is strongly influenced by the oral secretion from the insect, mode of feeding and degree of tissue damage at the feeding site and volatile signals from neighboring plants under herbivore attack (Walling, 2000). While most of the insect herbivores directly consume plant tissue, sucking and galling insects suck cell sap and manipulate normal plant anatomy and growth to develop a gall (Raman and Abrahamson, 1995, Raman, 2003). We have used large-scale “omics” technologies to assess the overall temporal host reprogramming during rice-gall midge interactions.

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Metabolomics, when applied to insect-plant interaction studies, can provide an insight into the network of metabolic pathways and a snapshot of metabolites that are believed to be endproducts of protein activity and thereby facilitating a better understanding of insect-plant interactions at the biochemical level. Different plant organs serve as sources of nutrition for the phytophagous insects. In such a scenario, the plants are forced to cater to the needs of insects while fulfilling their own metabolic requirements. Three hypothesis have been put forward (Price, 1991) to understand how insects would perform if the plants are under stress. According to plant stress hypothesis (PSH), insects perform better on the stressed hosts while the plant vigour hypothesis (PVH) states that insects will perform better on non-stressed hosts. Insect performance hypothesis (IPH) supports PVH by predicting that insects feeding on plant wood, sap or those that mine the plant organs, will perform better on stressed hosts while leaf-feeders and gall formers (having close association with their host) will perform better on non-stressed hosts (Price, 1991). It was found that the kind of stress the host plant faces also affects insect performance (Galway et al. 2004). Findings of a recent study by Agarrwal et al. (2014) suggested that PVH is applicable to rice-gall midge interaction since trehalose, a primary metabolite was identified as one of the infestation features during ricegall midge interaction. It was reported that these infestation features were common to both compatible and incompatible interactions and that the feeding larvae induce changes in the host’s metabolism such that the host remains healthy and non-stressed.

The Rice-gall midge incompatible interaction Rice-gall midge incompatible interaction is closer to plant-pathogen interaction rather than plant-insect interaction as it exhibits gene-for-gene interaction between plant R and insect avr genes (Bentur et al. 2003). Plants withstand pathogenic attacks by activating a large variety of defense mechanisms, including the HR (Alves et al. 2014). To date, majority of research in defense mechanism has been performed on dicotyledonous species such as tobacco and Arabidopsis, whereas in economically important cereal crops like rice, the mechanism of defense against insects is less understood (Kogel and Langen, 2005, Balmer et al. 2013). Pioneering research has been done on wheat-Hessian fly interactions (Mittapalli et al. 2006, Subramanyam et al. 2013) and now initiated in the case of rice-gall midge (Orseolia oryzae) interactions as well.

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Recently, using GC-MS-based metabolic profiling, several metabolites such as fatty acids, volatile alkenes, sterols, phenolic compounds were identified as resistance features during rice-gall midge incompatible interaction (Agarrwal et al. 2014). These resistance features were common to both types of incompatible interaction that either manifested with or without HR. These classes of compounds are known to play a role in plant defense against insects. The herbivore-induced volatile organic compounds (VOCs) are known to be involved in host defense against the herbivore by either deterring the insect or by attracting parasitoids of the insect (Ngumbi and Fadamiro, 2012).

Variation in gene expression in rice during HR+ type gall midge resistance HR+ type of incompatible interaction has been extensively studied in several plant-pest interactions. HR is characterized by rapid cell death that arises from elicitation of combined responses following R-avr gene recognition (Heath, 2000). Transcriptomics is an important tool to investigate the regulatory mechanisms of host-pest interactions since it can elucidate the modifications of metabolic and cellular routes due to the interaction. The molecular basis of HR+ type of rice-gall midge incompatible interaction was studied using suppressive subtraction hybridization (SSH) cDNA library that was enriched for differentially expressed transcripts, after gall midge biotype 4 (GMB4) infestation, in rice variety Suraksha (Rawat, 2012, Rawat et al. 2013).

We (Rawat et al. 2013) analyzed and explored the differential abundance of expressed sequence tags (ESTs) with various bioinformatics and statistics tools to identify system-wide transcriptome reprogramming events associated with HR+ type of resistance in Suraksha. We observed that most of the ESTs fell into five groups, mainly coding for hormone signaling; cell signaling; DNA structure and cell organization; secondary metabolism; and defense and stress related. Results showed that ESTs belonging to the secondary metabolism group and coding for phenylalanine ammonia lyase (PAL), carotenoids, terpenoids, lignin, mevalonate and non-mevalonate biosynthesis related enzymes and, ESTs, belonging to defense and stress-related group, coding for NBS-LRR (5 ESTs), NB-ARC (2 ESTs), pathogenesis related protein (OsPR10α; Oryza sativa pathogenesis related 10α) and other leucine rich repeat (LRR) family of proteins (7 ESTs) were abundant. NBS-LRRs are large abundant proteins involved in the detection of diverse pathogens, including bacteria, viruses, fungi, nematodes, insects and oomycetes (McHale et al. 2006). The putative candidate gene for Gm4 has been

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reported to encode an NBS-LRR domain containing protein while that of Gm2 (Himabindu, 2009) and gm3 encode NB-ARC domain containing proteins (Sundaram, 2007, Sama et al. 2014).

Further, RT-qPCR validation of shortlisted ESTs from the library suggested NBS-LRR, PAL (two ESTs each) and OsPR10α, as the most prominent orchestrators of the HR+ type resistance in Suraksha against gall midge. PAL is the rate-limiting step in the activation of phenylpropanoid pathway. Increased PAL activity is related with salicylic acid (SA) biosynthesis which is a local and systemic plant signaling molecule involved in defense response to pathogen attack (Milosevic and Slusarenko, 1996). We observed that all the 7 copies of PAL gene family present in rice genome, were up-regulated in Suraksha at 120 hai (hours after infestation) with GMB1 while at 24 hai only OsPAL1 was significantly upregulated which showed a substantial increase (~ 5.0 fold at 120 hai) (Rawat et al. unpublished data). We also observed strong induction of RSOsPR10α (root specific Oryza sativa pathogenesis related 10α) over 40 and 23-fold in Suraksha at 24 and 120 hai with GMB1, respectively (Rawat et al. 2010). It should be noted that both these genes were downregulated in Kavya rice variety that possesses HR- type of gall midge resistance (Rawat et al. 2012a). Our results indicated that HR+ type gall midge resistance in rice could be initiated by recognition of the unknown virulence factors from gall midge by the by the product of plant R genes, (possibly by NBS-LRR), which could further lead to an oxidative burst.

This study thus created a larger picture of genome-wide changes in expression and regulation of transcripts that provide an initial platform for R gene discovery and to elaborate the resistance mechanism in the Suraksha rice variety against the gall midge. It is well documented that response of plants against leaf feeding insects is akin to wounding and associated with jasmonic acid pathway while defense response against plant sap sucking insects like aphids, and galling insects, has been found to be associated with salicylic acid pathway (Walling, 2000, Gatehouse, 2002). Iwai et al. (2007) reported that topical application of SA triggers resistance to the blast fungus Magnaporthe oryzae in adult plants but not in young seedlings of rice. However, synthetic SA analogs such as probenazole and benzothiadiazole (BTH) are reported to induce defense responses in rice against a wide range of pathogens including Magnaporthe oryzae and bacterial leaf blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) (De Vleesschauwer et al. 2013, 2014, Xu et al. 2013). Our findings also indicate that HR+ type mechanism showed the involvement of SA mediated pathway 11

and secondary metabolites production during the interaction and shared more similarities with rice-pathogen interaction, rather than rice-insect interaction.

Variation in gene expression in rice during HR- type gall midge resistance HR- type of resistance in plants against pathogen is also known as extreme resistance (ER) which is activated by a number of R gene encoded proteins during Effector Triggered Immunity (ETI) (Bendahmane et al. 1999, Eggenberger et al. 2008, Wen et al. 2013). For instance, Rx locus in potato exhibits extreme resistance against potato virus X (PVX) and is not associated with host cell death (Bendahmane et al. 1999). In addition to Rx, RTM1/RTM2 in Arabidopsis (Whitham et al. 2000) and Ry in potato (Valkonen et al. 1994) are among other phenotypically unusual R genes that provide resistance against plant viruses without induction of HR at the inoculation site. Among plant-virus interactions, the N–avr Tobacco mosaic virus (TMV) and Rx–avr PVX (Moffett and Klessig, 2008) represent typical HR+ and ER, respectively. However, the relationship between ER and HR is not clear. ER expression is epistatic to that of HR in the Ryadg–Potato virus Yο (PVY) and Rx–avr PVX pathosystems (Valkonen et al. 1994, Bendahmane et al. 1999). Höglund et al. (2005) suggested that hypersensitivity and symptomless (HR- type) resistance are the two different components in another gall midge plant interaction (Salix-Dasineura interaction) and suspected an unknown mechanism of resistance in symptomless plants.

We used microarray technology to elucidate HR- type of gall midge resistance in Kavya rice variety against avirulent gall midge biotype 1 (GMB1) (Rawat 2012, Rawat et al. 2012a). We observed a small number of transcript modulation in HR- type incompatible interaction of Kavya-GMB1 when compared with the compatible interaction of Kavya with a virulent biotype of gall midge (GMB4M). This weak transcriptome response was in concordance with the tomato (Solanum lycopersicum)-powdery mildew fungus interaction (Fung et al. 2008). Similar observations were noticed in citrus- Huanglongbing (HLB) transcriptome, wherein only 17 genes were modulated in resistant genotype (US 897) in comparison to 326 genes that were significantly upregulated in the susceptible citrus genotype (Cleopatra) (Albrecht and Bowman, 2012). Of the 17 genes, the constitutive disease resistance 1 (CDR1) gene was highly expressed in resistant genotype (11.2 fold in uninfected US 897 as compared with 3.3 fold in uninfected Cleopatra).

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Microarray analysis

followed

by

functional

categorization

using

rice

database

(rapdb.dna.affrc.go.jp) and MapMan (Thimm et al. 2004) pathway analysis revealed that biotic stress related genes, cinnamoyl-CoA reductase and respiratory burst oxidase (RBO) genes such as AtRbohF and NADPH oxidase were abundant in HR- type of resistance found in Kavya (Rawat et al. 2012a). ROS is produced during biotic and abiotic stresses and it has been noted that AtrbohD and AtrbohF produce ROS that negatively regulate unrestricted cell death in lesion mimic mutant lsd1 of Arabidopsis (Torres et al. 2005). Höglund et al. (2005) also reported depletion of H2O2, a molecule required for ROS generation, in HR− type (symptomless resistance) while accumulation of H2O2 was noticed in HR+ type interaction during Salix–Dasineura interaction. AtRbohF is crucial in HR-related cell death regulating metabolomic responses and resistance (Chaouch et al. 2012).

We have isolated and characterized full length RSOsPR10α gene along its promoter region in TN1, Kavya and Suraskha (Rawat et al. 2010). Cis acting elements in the promoter regions were identified showing four putative mutations (PALBOXAPC, GT-1 binding sites, bZIP binding site and GATA binding site) in the promoter of RSOsPR10α allele in Kavya. Similar observations were made by Hwang et al. (2008) demonstrating that mutations in the cis acting sites in the promoter region of OsPR10a were responsible for lack of induction of the gene by salicylic acid. These evidences suggest that mutation in the promoter region of RSOsPR10α gene could be responsible for its lack of HR induction in Kavya against gall midge. Further alternate, novel, pathway of HR-independent resistance response in Kavya rice against gall midge GMB1 infestation was described by Rawat et al. (2010, 2012a).

Based on these observations, we proposed two hypotheses to explain HR- type of resistance in Kavya; first it could be due to the expression of a constitutive R gene in Kavya against gall midge and/or secondly, it could be a result of “extreme resistance” (ER). Evidence for involvement of a constitutive R gene could be explained as very few resistance-specific transcripts were identified through microarray analysis as the R gene product(s) was being constitutively expressed (at high or low abundance levels) and therefore, expected to be only weakly modulated in response to gall midge attack. The second hypothesis to explain this phenomenon is based on ER wherein the R gene product is sufficient to cease the pathogen without involving HR. These R gene products, which are probably expressed constitutively in Kavya, are sufficient to trigger a cascade of events that kill the insect without HR expression.

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Based on these observations we suggest that Gm1-conferred resistance in Kavya could be a mode of “extreme resistance” wherein the insect dies without requiring cell death in the host.

The rice-gall midge compatible interaction Earlier studies involving transcriptomic, metabolomic and proteomic analyses during plantpathogen interaction, focused mainly on resistance mechanism. In last few years, focus has been on the susceptibility mechanism and its importance. Compatible interaction between rice and gall midge is a phenomenon that does not resemble a situation in the plant-pathogen interaction wherein the plant dies upon pathogen attack. During rice-gall midge compatible interactions, when the plant fails to suppress the insect virulence it accommodates and localizes the feeding maggot, and is forced to provide it with nutrients but no plant mortality ensues. The gall midge larva manipulates the rice plant and creates a gall-like structure (nutritive tissue) for its growth and development (Bentur et al. 2003, Rawat et al. 2012b). It is also known that organisms such as bacterial and fungal parasites and several other insects induce plant gall formation (Meyer, 1987, Harris et al. 2003). To understand the susceptibility genes and their mode of function during compatible rice-gall midge interactions two techniques have been used namely, SSH library construction and microarray analysis. SSH library construction revealed up-regulated genes in the rice variety TN1 (having no R gene) during compatible interaction with GMB4M. In addition, microarray analysis identified several up- and down-regulated genes involved in the compatible interaction of Kavya with GMB4M (carrying ineffective Gm1 gene against GMB4M). Further, for the functional validation of these genes another genotype Suraksha (carrying ineffective Gm11 gene against GMB4M) was included in RT-PCR assays (Rawat et al. 2012b).

Screening of SSH libraries revealed a large number of ESTs related to primary metabolism, nutrient translocation and transporters-related genes to be up-regulated which most likely support the survival of the virulent maggots. Conversely, ESTs related to defense and secondary metabolism were down-regulated and therefore, probably, suppressed the resistance mechanism. ESTs representing substrate transporter, peptide transporter and amino acid transporters were also induced in compatible interactions suggesting that plants synthesize and supply their enriched nutrients to insects with help of these transporters, from site of synthesis to the site of insect feeding (Rawat et al. 2012b). The gall tissue or chamber can accumulate the nutrient compounds by breaking them down into small amides and peptides that are supplied from the host plant (Bronner, 1992). One of the sugar transporters 14

OsSWEET11, acts as a carrier of sugar molecules supplied to the pathogen Xanthomonas oryzae. By mutating this transporter the pathogen growth was reduced indicating that transporters were necessary for the pathogen growth and development (Chen, L.Q. et al. 2010). MADS box transcriptional factors have been found to be involved in plant growth, development and flower development. Approximately, 75 MADS have been identified in rice (Arora et al. 2007) whose roles are still unclear. MADS18 was found to be induced commonly in all three susceptible genotype at both the time points studied (24 and 120 hai) (Rawat et al. 2012b). Similarly, MADS26 has been reported to be involved in biotic and abiotic compatible interaction. After mutating this gene, rice plants were found to be resistant against Magnaporthe oryzae and Xanthomonas oryzae and also to drought stress. Overexpression of this gene led to susceptibility of host to the pathogen (Gantet et al. 2013).

Enhanced primary metabolism has been previously observed in growing nutritive tissue to meet the increased metabolic demands of the plant upon successful pathogen infection (Goethals et al. 2001). During compatible interactions between different rice genotypes and gall midge biotypes, genes involved in primary metabolism were commonly up-regulated while translationally controlled tumor protein (TCTP), an important component of Target of Rapamycin (TOR) signaling, was found to be commonly down-regulated (Rawat et al. 2012b). Moreover, TCTP was implicated as a candidate for susceptibility genes during ricegall midge interaction (Rawat et al. 2013). TOR signaling, was found to be involved in insect growth and molting (Kemirembe et al. 2012) and also in transduction of nutritional signals that regulate cell growth and metabolism (Howell and Manning, 2011, Siddle, 2012). Additionally, in yeast, glutamine starvation has been shown to affect a subset of TOR controlled transcription factor and TOR signaling pathway was reported to sense intracellular glutamine levels (Crespo et al. 2002). In our study involving GC-MS based metabolic profiling of host tissues, glutamine was reported as a susceptibility feature during rice-gall midge compatible interaction (Agarrwal et al. 2014). Glutamine is the amino acid that is utilized for de novo synthesis of amino acids in insects and mammals. The other amino acids that cannot be synthesized de novo by the insect, are called essential amino acids, and the feeding maggots fulfil their requirement by manipulating C/N shift in the host plant and utilize free essential amino acids necessary for their growth and development (Zhu et al. 2008). A few aphid species are also well known for manipulating nutrition from within the wheat plant by increasing the free amino acid pool (Telang et al. 1999, Eleftherianos et al. 2006). Similarly, five amino acids i.e. alanine (ala), aspartate (asp), glutamate (glu), glycine 15

(gly) and serine (ser) were found to accumulate in wheat-Hessian fly compatible interaction (Saltzmann et al. 2008).

Some genes were specifically induced in only one of the susceptible rice genotypes. As mentioned above, some of the defense, transcriptional factor and respiratory burst oxidase related genes such as LRR, NAC domain, NADPH oxidase and AtRbohF showed several fold up-regulation at 24 hai only in Kavya rice variety. This suggests that even though products of Gm1 are unable to recognize effectors from virulent biotype GMB4M, basal defense in Kavya against GMB4M is active in initial stages of infestation. In contrast, genes related to GRAS family transcriptional factor and transporter such as DELLA protein RGL1, CBS domain protein and ABC transporter were specifically down-regulated in Suraksha at 24 hai. These results indicated that Gm11 may be a weaker R gene against the virulent biotype as compared to Gm1 in Kavya.

From the above account it is clear that the host gene expression is significantly modified with the onset of insect attack. Depending on the resistant rice genotypes and the presence of gall midge resistance genes in them, it is apparent that the pest has evolved multiple pathways to suppress the host resistance. So much so, there is evidence that some rice varieties have opted for an ‘always on’ type of resistance (Rawat et al. 2012a) that would actually be a big burden on the plants in the long run in terms of energy use efficiency. However, these studies have brought in new insights into the understanding of the mode of resistance and the roles that the interaction plays in modulating resistance either in favor of the host or pest in their respective battle for survival.

Survival strategies adopted by the gall midge Insects have evolved tremendous survival ability (Misof et al. 2014) due to their adapting competence, large population size, extremely plastic and extensible anatomy. Phytophagous insects have intimate associations with their respective hosts since time immemorial (Smith and Clement, 2012). There is an ongoing strife between the host and the insect pest for survival. The outcome of this altercation favors the plant host when it evolves a strategy for survival against the insect and alternatively, swings towards the insect when it has carried out a successful invasion against the host. The success of the gall midge in overcoming the ricehost defense, in a compatible interaction, leads to completion of its life cycle while triggering alterations in the plants metabolic machinery. Extensive investigations to unravel the 16

molecular basis of virulence have revealed important features of this pest (Rawat et al. 2010, Sinha et al. 2011a, 2011b, 2012a, 2012b). These investigations have provided valuable clues revealing strategies adopted by the insect to survive and overcome the defense mechanism of the host. It has been suggested that apart from the evolution of different molecular mechanisms to evade the host defense, the insect’s sexual dimorphism and abnormal chromosome cycle (Sahu et al. 1996), characteristics that are linked to virulence, provides it with additional arsenal in the battle for survival.

Molecular mechanism: shield and strategy for host defense evasion or succession Feeding by midges on hosts includes initial injection of salivary proteins or compounds that possibly facilitates suppression of plant defense (Chen et al. 2004). The proteins of saliva include enzymes that have the ability to alter or reprogram host physiology in favor of the insect (Chen et al. 2006, 2008). These secreted salivary gland proteins (SSGPs) have been implicated to be effectors and are likely to be responsible for manipulation of host metabolic pathways so as to benefit the insect either in survival or feeding or both (Chen, M-S. et al. 2010). Our screening of cDNAs coding for SSGPs in the Asian rice gall midge identified genes coding for oligosaccharyl transferase (OST) and nucleoside diphosphate kinase (NDPK) (Sinha et al. 2011a, 2012a). NDPK from the gall midge was shown to be involved in compatible interactions and probably aided the insect in speeding the elongation of the host cells (hyperplasia), for providing them with better nourishment, and also speeding the production of the gall. Further analysis revealed that expression of NDPK was specifically higher in the salivary glands of maggots feeding on the susceptible host. The coleoptile elongation assay performed using recombinant NDPK suggested that the protein does indeed have cell elongation capabilities and, interestingly, E.coli cells expressing the gall midge NDPK showed higher salt tolerance. This indicated that NDPK has more than one role within the cell (Sinha et al. 2012a). Over-expression of transcripts encoding OST in salivary glands during initial maggot establishment in the susceptible plant hints at its involvement either in glycosylation of harmful proteins or to conform proteins to suit the maggot’s need (Sinha et al. 2011a).

In addition to salivary secretions, an important step towards winning the “arms race” is to improvise or invent strategies to avoid the defense response(s) or develop resistance against them. After successful penetration into the plant, the nature of the interaction (compatible or incompatible) is decided by the efficacy of strategies adopted by the insect to counter host 17

defense. To assess the changes in the insect upon infestation, the transcriptome of the rice gall midge maggots when feeding on a susceptible and a resistant host was sequenced (Sinha et al. 2012b). Roche 454 next generation sequencing (NGS) technique was employed to obtain the transcriptome data. This not only helped in generating a large dataset of genes expressed commonly in both compatible and incompatible interactions but also genes expressed exclusively in one of the two interactions.

Data mining of the transcriptome, led to the identification of genes involved in immunity, melanisation and signal transduction. These genes are probably necessary for the feeding maggots to overcome the defense response of the host plant. Also, transcripts related to genes involved in ROS pathways were prevalent. These probably helped in combating the ROS molecules generated by the plant in its defense against the maggots. Other than these, transcripts coding for proteases, protein kinases and genes involved in apoptosis were also commonly present. The high prevalence of these genes in the transcriptome reveals their significance in the rice-gall midge interaction (Sinha et al. 2012b). Further studies need to be conducted to completely understand their individual roles in the rice-gall midge interaction. Comparative analyses between transcripts represented in the maggots in the compatible or incompatible interaction revealed involvement of metabolic pathways such as ABC transporter, diterpenoid biosynthesis and Notch signaling in the incompatible interaction (Sinha et al. 2012b). In contrast, genes involved in amino acid biosynthesis pathways for valine, leucine and isoleucine were over-expressed in the compatible interaction. These amino acids are essential for sap feeders and a similar pattern was observed in the Hessian fly-wheat (Saltzmann et al. 2008) and the Russian wheat aphid-wheat interactions (Swanevelder et al. 2010, Anantakrishnan et al. 2014) as well. Maggots in compatible interaction coordinate effectively with the host plant metabolic machinery by diverting host metabolites for their own benefit.

Studies have also been conducted on individual genes to decipher their role in the rice-gall midge interaction. One such study by Sinha et al. (2011b) involved two serine-protease genes (OoProtI and OoProtII). Both the genes were significantly up-regulated in maggots feeding on a resistant host than on a susceptible host. Most plants that are frequently subjected to larval feeding produce proteinase inhibitors as defense molecules. These inhibitors cause starvation of insects due their inability to digest plant proteins, ultimately leading to insect mortality (Jongsma et al. 1995, Yang et al. 2009). To avoid this, insects produce enzymes 18

(serine-proteases) that degrade these inhibitors. Overproduction of these serine-proteases would lead to normal feeding by the insect and eventually lead to a compatible reaction (Brioschi et al. 2007).

Sexual dimorphism and abnormal chromosomal cycle in gall midge: an important armor in the battle for survival The Asian rice gall midge is a good example of a karyological sexual dimorphism. As in case of Hessian fly, female adults possess eight chromosomes (2n=8, two pairs of autosomes and two pairs of sex chormosomes) whereas in male somatic cells there are two pairs of autosomes and two monosomic sex chromosomes (2n=6; Sahu et al. 1996). In addition there is a large number of elimination of chromosomes in the germline cells. This is accomplished by selective elimination of paternal chromosomes during post-fertilization cell division. Thus, female insects carry more genetic material than their male counterparts suggesting prevalence of more number of genes in the females. Another interesting feature of this insect is the production of unisexual progenies i.e. a female produces either all-female or all-male progeny (Sain and Kalode, 1985). Few exceptional females produce offspring of both sexes but in unequal proportion and therefore, the female:male ratio is always biased towards female progenies. Further, involvement of bacteria (Wolbachia) that leads to sexual bias towards production of more females has also been suggested (Behura et al. 2001). Furthermore, this inclination towards female progeny and the role of endosymbionts, such as Wolbachia, in insect virulence need to be studied in depth.

Conclusions and future perspectives Deployment of naturally occurring resistance is probably the most environment friendly way of developing gall midge resistant rice varieties. However, as resistance in rice to gall midge is governed by a single dominant gene, the durability of any deployed resistance gene is likely to break down due to constant insect pressure and also due to the capacity of gall midges to quickly evolve new biotypes capable of breaking down deployed resistance genes. Therefore, it is a constant endeavour by plant breeders to identify and deploy relevant resistance genes to remain one, if not, several steps ahead of the insect pest. However, this was quite a tedious process in the past. But with the advent of new technologies and information being made available to the breeder, he/she is better informed and armed with more reliable data to enable the breeder to make a well-judged choice for breeding rice varieties with durable resistance to gall midge. Several transcriptomics-based studies provide 19

molecular data that elucidates the novel mechanisms of R genes identified in rice germ plasm. Breeders can thus resort to pyramiding two or more R genes with novel and contrasting mechanisms of resistance so that the break down of resistance, by newly evolving virulent biotypes, is delayed. Additionally, these studies will also provide a chance to better understand the gall midge resistance mechanism in rice. It would be interesting to study if rice mutants, lacking the susceptibility genes, are resistant to existing as well as newly evolving gall midge biotypes.

Recent metabolomics-based studies identified several resistance metabolites in the host that could be applied exogenously on susceptible hosts to study their effect in terms of gall midge maggot survival rate and subsequently for controlling the pest. Additionally some of the volatile compounds identified in the study, could be tested via electroantennography to study their capacity to function as chemical cues to attract gall midge adult flies during the egg laying process. A detailed evaluation of the transcriptome of the gall midge larvae, feeding on susceptible or resistant host, led to the identification of certain effectors of insect virulence. As an extension to these efforts, studies are under way to identify microRNAs that play a crucial role during rice-gall midge interaction. Once the targets for these RNAi inducing molecules are identified and validated, it would be interesting to develop an artificial diet for gall midge larvae supplemented with these molecules to test their efficacy in inducing larval mortality.

In conclusion, this review touches upon various important aspects of the interaction between rice host and the Asian rice gall midge. It represents details of various completed and ongoing studies, both at the host as well as pest levels. Important innovations and technologies developed in the recent past have been utilized to decipher the molecular basis of rice defense and gall midge counter-defense. We have enumerated diverse strategies adopted by the host as well as the gall midge, aimed at ensuring their respective survival, and also highlighted the lacunae in the current understanding of this important interaction. Although, more insights have been obtained by the molecular studies carried out so far, the future awaits the development of more durable and dependable source of resistance in rice to be able to control gall midge, a major pest of an economically important cereal crop of the world.

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Acknowledgements Research on rice gall midge and gall midge-rice interaction in SN’s laboratory is partially supported by core grants from ICGEB and extra-mural funding from the Department of Biotechnology (DBT) and the Indian Council of Agricultural Research (ICAR), Government of India. JSB wishes to acknowledge extra mural funding from DBT and ICAR. RA and IA thank the University Grants Commission (UGC), Government of India, for Senior Research Fellowships.

Supplementary figure legend Supplementary Fig. 1. The first instar larva of the Asian rice gall midge showing sternal spatula (arrow).

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References: Agarrwal, R., Bentur, J.S., Nair, S., 2014. Gas chromatography mass spectrometry based metabolic profiling reveals biomarkers involved in rice‐gall midge interactions. Journal of Integrative Plant Biology, 56, 837-848.

Albrecht, U., Bowman, K. D., 2012. Transcriptional response of susceptible and tolerant citrus to infection with Candidatus Liberibacter asiaticus. Plant Science, 185-186, 118-130.

Alves, M. S., Dadalto, S. P., Gonçalves, A. B., de Souza, G. B., Barros, V. A., Fietto, L. G., 2014. Transcription factor functional protein-protein interactions in plant defense responses. Proteomes, 2, 85-106.

Anathakrishnan, R., Sinha, D.K., Murugan, M., Zhu, K.Y., Chen, M-S., Zhu, Y.C., Smith, C.M., 2014. Comparative gut transcriptome analysis reveals differences between virulent and avirulent Russian wheat aphids, Diuraphis noxia. Arthropod-Plant Interactions, 8, 79-88.

Andow, D.A., Bentur, J.S., 2010. Pedigreed crosses to estimate recessive virulence allele frequencies in natural populations of gall midges. Entomologia Experimentalis et Applicata, 135, 18-36.

Arora, R., Agarwal, P., Ray, S., Singh, A.K., Singh, V.P., Tyagi, A.K., Kapoor, S., 2007. MADS-box gene family in rice: genome-wide identification, organization and expression profiling during reproductive development and stress. BMC Genomics, 8, 242.

Balmer, D., Planchamp, C., Mauch-Mani, B., 2013. On the move: induced resistance in monocots. Journal of Experimental Botany, 64, 1249-1261 Behura, S.K., Nair, S., Sahu, S.C., Mohan, M., 2000. An AFLP marker that differentiates biotypes of the Asian rice gall midge (Orseolia oryzae, Wood-Mason) is sex-linked and also linked to avirulence. Molecular Genomics and Genetics, 263, 328-334.

22

Behura, S.K., Sahu, S.C., Mohan, M., Nair, S., 2001. Wolbachia in the Asian rice gall midge, Orseolia oryzae (Wood-Mason): correlation between host mitotypes and infection status. Insect Molecular Biology, 2, 163-171.

Bendahmane, A., Kanyuka, K., Baulcombe, D.C., 1999. The Rx gene from potato controls separate virus resistance and cell death responses. Plant Cell, 11, 781-791.

Bentur, J.S., Kalode, M.B., 1996. Hypersensitive reaction and induced resistance in rice against Asian rice gall midge Orseolia oryzae. Entomologia Experimentalis et Applicata, 78, 77-81.

Bentur, J.S., Pasalu, I.C., Kalode, M.B., 1992. Inheritance of virulence in rice gall midge, Orseolia oryzae. Indian Journal of Agricultural Sciences, 62, 492-493.

Bentur, J.S., Pasalu, I.C., Sarma, N.P., Prasada Rao, U., Mishra, B., 2003. Gall midge resistance in rice, Directorate of Rice Research, Hyderabad. Bentur, J.S., Cheralu, C., Rao, P.R.M., 2008. Monitoring virulence in Asian rice gall midge populations in India. Entomologia Experimentalis et Applicata, 129, 96-106.

Bentur, J.S., Padmakumari, A.P., Jhansi Lakshmi, V., Padmavathi, Ch., Kondala Rao, Y., Amudhan, S., Pasalu, I.C., 2011. Insect resistance in rice. Technical Bulletin, 51, Directorate of Rice Research, Hyderabad, India, pp. 85.

Bentur, J.S., Rawat, N., Sinha, D.K., Nagaraju, J., Nair, S., 2013. New genetic avenues for insect pest management in rice as revealed by studies on gall midge, in: Muralidharan, K., Siddiq, E.A., (Eds.), International Dialogue on Perception and Prospects of Designer Rice. Society for Advancement of Rice Research, Directorate of Rice Research, Hyderabad, India, pp. 185-187.

Brioschi, D., Nadalini, L.D., Bengtson, M.H., Sogayar, M.C., Moura, D.S., Silva-Filho, M.C., 2007. General upregulation of Spodoptera frugiperda trypsins and chymotrypsins allows its adaptation to soybean proteinase inhibitor. Insect Biochemistry and Molecular Biology, 37, 1283-1290. 23

Bronner, R., 1992. The role of nutritive cells in the nutrition of cynipids and cecidomyiids, in: Shorthouse, J.D., Rohfritsch, O., (Eds.), Biology of insect-induced galls. Oxford University Press, Oxford, pp. 118-140. Chaouch, S., Queval, G., Noctor, G., 2012. AtrRbohF is a crucial modulator of defenseassociated metabolism and a key factor in the interplay between intracellular oxidative stress and pathogenesis responses in Arabidopsis. The Plant Journal, 69, 613-627.

Chaudhary, B.P., Srivastava, P.S., Shrivastava, M.N., Khush, G.S., 1985. Inheritance of resistance to gall midge in some cultivars of rice. in: Rice Genetics, International Rice Research Institute, Manila, Philippines, pp. 523-528.

Chen, L.Q., Hou, B.H., Lalonde, S., Takanaga, H., Hartung, M.L., Qu, X.Q., Guo, W.J., Kim, J.G., Underwood, W., Chaudhuri, B., Chermak, D., Antony, G., White, F.F., Somerville, S.C., Mudgett, M.B., Frommer, W.B., 2010. Sugar transporters for intercellular exchange and nutrition of pathogens. Nature, 468, 527-532.

Chen, M-S., Fellers, J.P., Stuart, J.J., Reese, J.C., Liu, X.M., 2004. A group of related cDNAs encoding secreted proteins from Hessian fly [Mayetiola destructor (Say)] salivary glands. Insect Molecular Biology, 13, 101-108. Chen, M-S., Fellers, J.P., Zhu, Y.C., Stuart, J.J., Hulbert, S., El Bouhssini, M., Liu, X., 2006. A super-family of genes coding for secreted salivary gland proteins from the Hessian fly, Mayetiola destructor. Journal of Insect Science, 6, 1-13.

Chen, M-S., Zhao, H.X., Zhu, Y.C., Scheffler, B., Liu, X., Liu, X., Hulbert, S., Stuart, J.J., 2008. Analysis of transcripts and proteins expressed in the salivary glands of Hessian fly (Mayetiola destructor) larvae. Journal of Insect Physiology, 54, 1-16.

Chen, M-S., Liu, X., Yang, Z., Zha, H., Shukle, R.H., Stuart, J.J., Hulbert, S., 2010. Unusual conservation among genes encoding small secreted salivary gland proteins from a gall midge. BMC Evolutionary Biology, 10, 296.

24

Cheng, X., Zhu, L., He, G., 2013. Towards understanding of molecular interactions between rice and the brown planthopper. Molecular Plant, 6, 621-634.

Crespo, J.L., Powers, T., Fowler, B., Hall, M.N., 2002. The TOR-controlled transcription & activators GLN3, RTG1, and RTG3 are regulated in response to intracellular levels of glutamine. Proceedings of the National Academy of Sciences USA, 99, 6784-6789.

De Vleesschauwer, D., Gheysen, G., Höfte, M., 2013. Hormone defense networking in rice: tales from a different world. Trends in Plant Science, 18, 555–565.

De Vleesschauwer, D., Xu, J., Höfte, M., 2014. Making sense of hormone-mediated defense networking: from rice to Arabidopsis. Frontiers in Plant Science, 5, 611.

DeYoung, B.J., Innes, R.W., 2006. Plant NBS-LRR proteins in pathogen sensing and host defense. Nature Immunology, 7, 1243-1249.

Divya, D., Himabindu, K., Nair, S., Bentur, J.S., 2015. Cloning of a gene encoding LRR protein and its validation as candidate gall midge resistance gene, Gm4, in rice. Euphytica, 203, 185-195.

Du, B., Zhang, W.L., Liu, B.F., Hu, J., Wei, Z., Shi, Z.Y., He, R.F., Zhu, L.L., Chen, R.Z., Han, B., He, G., 2009. Identification and characterization of Bph14, a gene conferring resistance to brown planthopper in rice. Proceedings of the National Academy of Sciences USA, 106, 22163-22168.

Dutta, S.S., Divya, D., Durga Rani, Ch.V., Dayakar Reddy, T., Visalakshmi, V., Cheralu, C., Ibohal Singh, Kh., Bentur, J.S., 2014. Characterization of gall midge resistant rice genotypes using resistance gene specific markers. Journal of Experimental Biology and Agricultural Sciences, 2, 439-446.

Eggenberger, A. L., Hajimorad, M. R., Hill, J. H., 2008. Gain of virulence on Rsv1-genotype soybean by an avirulent Soybean mosaic virus requires concurrent mutations in both P3 and HC-Pro. Molecular Plant-Microbe Interactions, 21, 931-936.

25

Eleftherianos, I., Vamvatsikos, P., Ward, D., Gravanis, F., 2006. Changes in the levels of plant total phenols and free amino acids induced by two cereal aphids and effects on aphid fecundity. Journal of Applied Entomology, 130, 15-19.

Fujita, D., Kohli, A., Horgan, F.G., 2013. Rice resistance to planthoppers and leafhoppers. Critical Reviews in Plant Science, 32, 162-191.

Fung, R.W.M., Gonzalo, M., Fekete, C., Kovacs, L.G., He, Y., Marsh, E., McIntyre, L.M., Schachtman, D.P., Qiu, W., 2008. Powdery mildew induces defense-oriented reprogramming of the transcriptome in a susceptible but not in a resistant grapevine. Plant Physiology, 146, 236-249.

Galway, K.E., Duncan, R.P., Syrett, P., Emberson, R.M., Sheppard, A.W., 2004. Insect performance and host-plant stress: A review from a biological control perspective, in: Cullen, J.M., Briese, D.T., Kriticos, D.J., Lonsdale, W.M., Morin, L., Scott, J.K., (Eds.), Proceedings of the XI International Symposium on Biological Control of Weeds: Canberra, Australia, 27 April - 2 May 2003, Canberra, Australia: CSIRO Entomology, pp. 394-399.

Gantet, P.A., Guiderdoni, E.A., Khong, N.A., Morel, J.B., 2013. Stress-resistant plants and their production. E.P. Patent 2635104 A1.

Gatehouse, J.A., 2002. Plant resistance towards insect herbivores: a dynamic interaction. New Phytologist, 156, 145-169.

Goethals, K., Vereecke, D., Jaziri, M., Van Montagu, M., Holsters, M., 2001. Leafy gall formation by Rhodococcus fascians. Annual Review of Phytopathology, 39, 27-52.

Harris, M.O., Stuart, J.J., Mohan, M., Nair, S., Lamb, R.J., Rohfritsch, O., 2003. Grasses and gall midges: Plant defense and insect adaptation. Annual Review of Entomology, 48, 549577.

Harris, M.O., Freeman, T.P., Rohfritsch, O., Anderson, K.G., Payne, S.A., Moore, J.A., 2006. Virulent Hessian fly (Diptera: Cecidomyiidae) larvae induce a nutritive tissue during

26

compatible interactions with wheat. Annals of the Entomological Society of America, 99, 305-316.

Heath, M.C., 2000. Hypersensitive response-related death. Plant Molecular Biology, 44, 321334. Heinrichs, E.A., 1994. Host plant resistance, in: Heinrichs (Eds.) Biology and management of rice insects. Wiley Eastern Limited, New Delhi, pp. 517-547.

Heinrichs, E.A., Medrano, F.G., Rapusas, H.R., 1985. Genetic evaluation for insect resistance. International Rice Research Institute, Manila, pp. 336.

Himabindu, K., 2009. Identification, tagging and mapping of rice gall midge resistance genes using microsatellite markers, PhD thesis, Acharya Nagarjuna University, Hyderabad, India.

Himabindu, K., Suneetha, K., Sama, V.S.A.K., Bentur, J.S., 2010. A new rice gall midge resistance gene in the breeding line CR57-MR1523, mapping with flanking markers and development of NILs. Euphytica, 174, 179-187.

Höglund, S., Larsson, S., Wingsle, G., 2005. Both hypersensitive and non-hypersensitive responses are associated with resistance in Salix viminalis against the gall midge Dasineura marginemtorquens. Journal of Experimental Botany, 56, 3215-3222.

Horgan, F.G., 2009. Mechanism of resistance: a major gap in understanding planthopper rice interactions, in: Heong, K.L., Hardy, B. (Eds.) Planthoppers: new threats to the sustainability of intensive rice production systems in Asia. International Rice Research Institute, Manila, pp. 281-302.

Howell, J.J., Manning, B.D., 2011. mTOR couples cellular nutrient sensing to organismal metabolic homeostasis. Trends in Endocrinology and Metabolism, 22, 94-102.

Hwang, S.H., Lee, I.A., Yie, S.W., Hwang, D.J., 2008. Identification of an OsPR10a promoter region responsive to salicylic acid. Planta, 227, 1141-1150.

27

Iwai, T., Seo, S., Mitsuhara, I., Ohashi, Y., 2007. Probenazole-induced accumulation of salicylic acid confers resistance to Magnaporthe grisea in adult rice plants. Plant and Cell Physiology, 48, 915–924.

Jongsma, M.A., Bakker, P.L., Peters, J., Bosch, D., Stiekema, W.J., 1995. Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of gut proteinase activity insensitive to inhibition. Proceedings of the National Academy of Sciences USA, 92, 80418045.

Kemirembe, K., Liebmann, K., Bootes, A., Smith, W.A., Suzuki, Y., 2012. Amino acids and TOR signaling promote prothoracic gland growth and the initiation of larval molts in the tobacco hornworm Manduca sexta. PLoS ONE, 7, e44429.

Khan, M.Q., Murthy, D.V., 1955. Some notes on the rice gall fly, Pachydiplosis oryzae (W.M.). Journal of the Bombay Natural History Society, 53, 97-102.

Khan, Z.R., Litsinger, J.A., Barrion, A.T., Villanueva, F.F.D., Fernandez, N.J. Taylo, L.D., 1991. World bibilography of rice stem borers 1794-1990. International Rice Research Institute and International Center of Insect Physiology and Ecology, pp. 792.

Kogel, K.H., Langen, G., 2005. Induced disease resistance and gene expression in cereals. Cellular Microbiology, 7, 1555–1564.

Kumar, A., Shrivastava, M.N., Shukla, B.C., 1998a. Inheritance and allelic relationship of gall midge biotype-1 resistance gene(s) in some new donors. Oryza, 35, 70-73.

Kumar, A., Shrivastava, M.N., Sahu, R.K., 1998b. Genetic analysis of ARC5984 for gall midge resistance-a reconsideration. Rice Genetics Newsletter, 15, 142-143. Kumar, A., Shrivastava, M.N., Shukla, B.C., 1999. A new gene for resistance to gall midge in rice cultivar RP2333-156-8. Rice Genetics Newsletter, 16, 85-87.

Kumar, A., Bhandarkar, S., Pophlay, D.J., Shrivastava, M.N., 2000. A new gene for gall midge resistance in rice accession Jhitpiti. Rice Genetics Newsletter, 17, 83-84. 28

Kumar, A., Jain, A., Sahu, R.K., Shrivastava, M.N., Nair, S., Mohan, M., 2005. Genetic analysis of resistance genes for the Rice Gall Midge in two rice genotypes. Crop Science, 45, 1631-1635. McHale, L., Tan, X., Koehl, P., Michelmore, R., 2006. Plant NBS-LRR proteins: adaptable guards. Genome Biology, 7, 212.

Meyer, J., 1987. Plant galls and gall inducers. Stuttgart, Gebruder Borntrager, pp. 291.

Misof, B., Liu, S., Meusemann, K., Peters, R.S., Donath, A., Mayer, C., Frandsen, P.B., Ware, J., Flouri, T., Beutel, R.G. et al., 2014. Phylogenomics resolves the timing and pattern of insect evolution. Science, 7, 763-767.

Milosevic, N., Slusarenko, A.J., 1996. Active oxygen metabolism and lignification in the hypersensitive response in bean. Physiological and Molecular Plant Pathology, 49, 143-158. Mittapalli, O., Shukle, R., Sardesai, N., Giovanini, M.P., Williams, C.E., 2006. Expression patterns of antibacterial genes in the Hessian fly. Journal of Insect Physiology, 52, 11431152.

Moffett, P., Klessig, D.F., 2008. Plant resistance to viruses: Natural resistance associated with dominant genes. Encyclopaedia of Virology, 3rd Edition.Mahy, B., Regenmortel, M.V., Elsevier Academic Press, Oxford.

Mohan, M., Nair, S., Bentur, J.S., Prasada Rao, U., Bennett, J., 1994. RFLP and RAPD mapping of rice Gm2 gene that confers resistance to biotype 1 of gall midge (Orseolia oryzae). Theoretical and Applied Genetics, 87, 782-788. Nair, S., Bentur, J.S., Prasada Rao, U., Mohan, M., 1995. DNA markers tightly linked to gall midge resistance gene (Gm2) are potentially useful for marker-aided selection in rice breeding. Theoretical and Applied Genetics, 91, 68-73.

29

Nair, S., Bentur, J.S., Sama, V.S.A.K., 2011. Mapping gall midge resistance genes: Towards durable resistance through gene pyramiding, in: Muralidharan, K., Siddiq, E.A., (Eds.), Genomics and Crop Improvement: Relevance and Reservations. Institute of Biotechnology, ANGR Agricultural University, Hyderabad, pp. 256-264. Ngumbi, E., Fadamiro, H., 2012. Species and sexual differences in behavioural responses of a specialist and generalist parasitoid species to host related volatiles. Bulletin of Entomological Research, 102, 710-718.

Price, P.W., 1991. The plant vigor hypothesis and herbivore attack. Oikos, 62, 244-251.

Raman, A., 2003. Cecidogenetic behaviour of some gall-inducing thrips, psyllids, coccids, and gall midges, and morphogenesis of their galls. Oriental Insects, 37, 359-413.

Raman, A., Abrahamson, W.G., 1995. Morphometric relationships and energy allocation in the apical rosette galls of Solidago altissima (Asteraceae) induced by Rhopalomyia solidaginis (Diptera: Cecidomyiidae). Environmental Entomology, 24, 635- 639.

Rawat, N., 2012. Identification and characterization of differentially expressed genes in rice involved in rice-gall midge interactions. PhD thesis, Osmania University, Hyderabad, India.

Rawat, N., Sinha, D.K., Rajendrakumar, P., Srivastava, P., Neeraja, C.N., Sundaram, R.M., Nair, S., Bentur, J.S., 2010. Role of pathogenesis related genes in rice gall midge interactions. Current Science, 99, 1361-1368.

Rawat, N., Neeraja, C.N., Sundaram, R.M., Nair, S., Bentur, J.S., 2012a. A novel mechanism of gall midge resistance in the rice variety Kavya revealed by microarray analysis. Functional & Integrative Genomics, 12, 249-264. Rawat, N., Neeraja, C.N., Nair, S., Bentur, J.S., 2012b. Differential gene expression in gall midge susceptible rice genotypes revealed by suppressive subtraction hybridization (SSH) cDNA libraries and microarray analysis. Rice, 5, 8.

30

Rawat, N., Himabindu, K., Neeraja, C.N., Nair, S., Bentur, J.S., 2013. Suppressive subtraction hybridization reveals that rice gall midge attack elicits plant-pathogen-like responses in rice. Plant Physiology and Biochemistry, 63, 122-130.

Reddy, V.A., Siddiq, E.A., Prasada Rao, U.P., Bentur, J.S., 1997. Genetics of resistance to rice gall midge (Orseolia oryzae). Indian Journal of Genetics, 57, 361-372.

Sahu, S.C., Bose, I.K., Pani, J., Rajamani, S., Mathur, K.C., 1996. Karyotype of rice gall midge, Orseolia oryzae (Wood-Mason). Current Science, 70, 874.

Sain, M., 1988. Studies on varietal resistance and bionomics of rice gall midge, Orseolia oyzae (Wood-Mason). PhD thesis. Osmania University, Hyderabad India. pp. 239.

Sain, M., Kalode, M.B., 1985. Traps to monitor gall midge population in rice. Current Science, 54, 876-877.

Saltzmann, K.D., Giovanini, M.P., Zheng, C., Williams, C.E., 2008. Virulent Hessian fly larvae manipulate the free amino acid content of host wheat plants. Journal of Chemical Ecology, 11, 1401-1410.

Sama, V.S.A.K., Rawat, N., Sundaram, R.M., Himabindu, K., Naik, B.S., Viratamath, B.C., Bentur, J.S., 2014. A putative candidate for the recessive gall midge resistance gene gm3 in rice identified and validated. Theoretical and Applied Genetics, 127, 113-124.

Shrivastava, M.N., Kumar, A., Bhandarkar, S., Shukla, B.C., Agrawal, K.C., 2003. A new gene for resistance in rice to Asian rice gall midge (Orseolia oryzae Wood Mason) biotype 1 population at Raipur, India. Euphytica, 130, 143-145.

Siddle, K., 2012. Molecular basis of signaling specificity of insulin and IGF receptors: neglected corners and recent advances. Frontiers in Endocrinology, 3, 1-24.

Sinha, D.K., Bentur, J.S., Nair, S., 2011a. Compatible interaction with its rice host leads to enhanced expression of the gamma subunit of oligosaccharyl transferase in the Asian rice gall midge, Orseolia oryzae. Insect Molecular Biology, 20, 567-575. 31

Sinha, D.K., Lakshmi, M., Anuradha, G., Rahman, S.J., Siddiq, E.A., Bentur, J.S., Nair, S., 2011b. Serine proteases-like genes in the rice gall midge show differential expression in compatible and incompatible interactions with rice. International Journal of Molecular Sciences, 12, 2842-2852. Sinha, D.K., Atray, I., Bentur, J.S., Nair, S., 2012a. Expression of Orseolia oryzae nucleoside diphosphate kinase (OoNDPK) is enhanced in rice gall midge feeding on susceptible rice hosts and its over-expression leads to salt tolerance in Escherichia coli. Insect Molecular Biology, 6, 593-603.

Sinha, D.K., Nagaraju, J., Tomar, A., Bentur, J.S., Nair, S., 2012b. Pyrosequencing-based transcriptome analysis of the Asian rice gall midge reveals differential response during compatible and incompatible interaction, International Journal of Molecular Sciences, 13, 13079-13103.

Smith, C.M., Clement, S.L., 2012. Molecular bases of plant resistance to arthropods. Annual Review of Entomology, 57, 309-328.

Srivastava, M.N., Kumar, A., Shrivastava, S.K., Sahu, R.K., 1993. A new gene for resistance to gall midge in rice variety Abhaya. Rice Genetics Newsletter, 10, 79-80. Stuart, J.J., Chen, M-S., Shukle, R., Harris, M.O., 2012. Gall midges (Hessian flies) as plant pathogens. Annual Review of Phytopathology, 50, 339-357.

Subramanyam, S., Zheng, C., Shukle, J.T., Christie, E., Williams, C.E., 2013. Hessian fly larval attack triggers elevated expression of disease resistance dirigent-like protein-encoding gene, HfrDrd, in resistant wheat. Arthropod-Plant Interaction, 7, 389-402. Sundaram, R.M., 2007. Fine mapping of rice gall midge resistance genes Gm1 and Gm2 and validation of the linked markers. PhD thesis. University of Hyderabad, Hyderabad, India.

32

Swanevelder, Z.H., Surridge, A.K., Venter, E., Botha, A.M., 2010. Limited endosymbiont variation in Diuraphis noxia (Hemiptera: Aphididae) biotypes from the United States and South Africa. Journal of Economic Entomology, 3, 887-897.

Tan, Y., Pan, Y., Zhang, Y., Lixia, Z., Xu, Y., 1993. Resistance to gall midge (GM) Orseolia oryzae in Chinese rice varieties compared with varieties from other countries. International Rice Research Newsletter, 18, 13-14.

Telang, A., Sandström, J., Dyreson, E., Moran, N.A., 1999. Feeding damage by Diuraphis noxia results in nutritionally enhanced phloem diet. Entomologia Experimentalis et Applicata, 91, 403-412.

Thimm, O., Blasing, O., Gibon, Y., Nagel, A., Meyer, S., Kruger, P., Selbig, J., Muller, L.A., Rhee, S.Y., Stitt, M., 2004. MAPMAN: A user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes. The Plant Journal, 37, 914939. Torres, M.A., Jones, J.D., Dangl, J.L., 2005. Pathogen-induced, NADPH oxidase-derived reactive oxygen intermediates suppress spread of cell death in Arabidopsis thaliana. Nature Genetics, 37, 1130-1134.

Valkonen, J.P.T., Slack, S.A., Plaisted, R.L., Watanabe, K., 1994. Extreme resistance is epistatic to hypersensitive resistance to potato virus Yο in a Solanum tuberosum subsp. andigena-derived potato genotype. Plant Disease, 78, 1177-1180.

Vijaya Lakshmi, P., Amudhan, S., Himabindu, K., Cheralu, C., Bentur, J.S., 2006. A new biotype of the Asian rice gall midge Orseolia oryzae (Diptera: Cecidomyiidae) characterized from the Warangal population in Andhra Pradesh, India. International Journal of Tropical Insect Science, 26, 207-211.

Walling, L.L., 2000. The myriad plant responses to herbivores. Journal of Plant Growth Regulation, 19, 195-216.

33

Wang, Y., Cao, L., Zhang, Y., Cao, C., Liu, F., Huang, F., Qiu, Y., Li, R., Lou, X., 2015. Map-based cloning and characterization of BPH29, a B3 domain-containing recessive gene conferring brown planthopper resistance in rice. Journal of Experimental Botany, doi:10.1093/jxb/erv318. War, A.R., Paulraj, M.G., Ahmad,T., Buhroo, A.A., Hussain, B., Ignacimuthu, S., Sharma, H.C., 2012. Mechanisms of plant defense against insect herbivores. Plant Signaling & Behavior, 7, 1306–1320.

Wen, R.H., Khatabi, B., Ashfield, T., Maroof, M.S., Hajimorad, M.R., 2013. The HC-Pro and P3 cistrons of an avirulent Soybean mosaic virus are recognized by different resistance genes at the complex Rsv1 locus. Molecular Plant-Microbe Interactions, 26, 203-215.

Whitham, S.A., Anderberg, R.J., Chisholm, S.T., Carrington, J.C., 2000. Arabidopsis RTM2 gene is necessary for specific restriction of tobacco etch virus and encodes an unusual small heat shock-like protein. Plant Cell, 12, 569-582. Xu, J., Audenaert, K., Höfte, M., De Vleesschauwer, D., 2013. Abscisic acid promotes susceptibility to the rice leaf blight pathogen Xanthomonas oryzae pv. oryzae by suppressing salicylic acid-mediated defenses. PLoS ONE, 8, e67413.

Yang, L., Fang, Z., Dicke, M., van Loon, J.J., Jongsma, M.A., 2009. The diamondback moth, Plutella xylostella, specifically inactivates Mustard Trypsin inhibitor 2 (MTI2) to overcome host plant defense. Insect Biochemistry and Molecular Biology, 39, 55-61.

Yasala, A.K., Rawat, N., Sama, V.S.A.K, Himabindu, K., Sundaram, R.M., Bentur, J.S., 2012. Analysis for gene content in rice genomic regions mapped for the gall midge resistance genes. Plant Omics Journal, 5, 405-413. Zhu, L., Liu, X.M., Liu, X., Jeannotte, R., Reese, J.C., Harris, M., Stuart, J.J., Chen, M-S., 2008. Hessian fly (Mayetiola destructor) attack causes dramatic shift in carbon/nitrogen metabolism in wheat. Molecular Plant-Microbe Interaction, 21, 70-78.

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Table 1: Gall midge resistance genes in rice, their chromosomal location, linked markers and spectrum of resistance across biotypes Group1

Rice Genotype

Resistance

Chr.

HR

Candi

Linked/

Gene

No.

type

date

specific 2

gene 1 2

W1263 Phalguna

Gm1 Gm2

9 4

+

2 2 2 2 2 3 4

Gm5 Gm6 Gm7 Gm9 Gm10 Gm11 gm3

? 4 4 ? ? 12 4

+ + + + + + +

4

ARC5984 Dukong # 1 RP2223 Madhuri L9 BG308 Suraksha RP2068-18-35 Abhaya

Gm4

8

+

4 5

Jhitpiti/Agani TN1

Gm8 None

8 -

NA

1

GMB1

GMB2

GMB3

+ +

R R

S R

R S

+ + + + +

R R R R R R R

R R R R R R R

+

R

+ NA

R S

marker

GMB4

Reference

GMB5

GMB6

GMB4M

S S

R R

R S

S S

Reddy et al. 1997 Mohan et al. 1994

R R R R R R R

S S S S S R R

R R R R R S R

S S S S S S S

S S S S S S R

Kumar et al. 1998b Tan et al. 1993 Kumar et al. 1999 Shrivasatava et al. 2003 Kumar et al. 2005 Himabindu et al. 2010 Kumar et al. 1998a

R

R

R

S

S

R

Srivastava et al. 1993

R S

R S

R S

S S

S S

R S

Kumar et al. 2000 -

3

Groups are based on the spectrum of resistance conferred by the gene across gall midge biotype

2 †

3

? NBARC† ? ? ? ? ? ? NBARC† NBLRR‡ PRP* None

Reaction against the gall midge biotype

Sama et al. 2014; ‡Divya et al. 2015; *Dutta et al. 2014; (?)/ Divya et al. Unpublished

Linked flanking or gene specific functional markers known (+) (Nair et al. 2011) or unknown (-); NA – not applicable;

HR Hypersensitive reaction GMB Gall midge biotype R: resistant; S: susceptible; Chr: Rice chromosome number ? Not determined (After Bentur et al. 2011)

35

•

Molecular studies reveal rice defence and gall midge counter-defence

•

Candidate R gene conferring gall midge resistance in rice functionally validated

•

Transcriptomics highlight massive reprogramming of host gene expression

•

Metabolomics reveal potential biomarkers for rice-gall midge interaction

•

Gall midge secretory salivary gland proteins influence host physiology

36

INSECT SECRETORY SALIVARY GLAND PROTEINS

HOST TRANCRIPTOMICS

HOST METABOLOMICS

RICE GALL MIDGE INTERACTION

INSECT TRANCRIPTOMICS

37