Integrated transcriptome and metabolite profiling highlights the role of benzoxazinoids in wheat resistance against Fusarium crown rot

Fusarium crown rot(FCR), caused by Fusarium spp., is a chronic and severe plant disease worldwide. In the last years, the incidence and severity of FCR in China has increased to the point that it is now considered a threat to local wheat crops. In this study, for the first time, the metabolites and transcripts responsive to FCR infection in the partial resistant wheat cultivar 04 Zhong 36(04 z36) and susceptible cultivar Xinmai 26(XM) were investigated and compared at 20 and 25 days post inoculation(dpi). A total of 443 metabolites were detected, of which 102 were significantly changed because of pathogen colonization.Most of these 102 metabolites belonged to the flavonoid, phenolic acid, amino acid and derivative classes.Some metabolites, such as proline betaine, lauric acid, ribitol, and arabitol, were stably induced by Fusarium pseudograminearum(Fp) infection at two time points and may have important roles in FCR resistance. In line with the reduced seedling height of 04 z36 and XM plants, RNA-seq analysis revealed that FCR infection significantly affected the photosynthesis activities in two cultivars. Furthermore, 15 jasmonate ZIM-domain genes(JAZ) in the significantly enriched ‘regulation of jasmonic acid mediated signaling pathway’ in 04 z36 were down-regulated. The down-regulation of these JAZ genes in 04 z36 may cause a strong activation of the jasmonate signaling pathway. Based on combined data from gene expression and metabolite profiles, two metabolites, benzoxazolin-2-one(BOA) and 6-methoxy-benzoxazolin-2-one(MBOA), involved in the benzoxazinoid-biosynthesis pathway, were tested for their effects on FCR resistance. Both BOA and MBOA significantly reduced fungal growth in vitro and in vivo, and, thus, a higher content of BOA and MBOA in 04 z36 may contribute to FCR resistance. Above all, the current analysis extends our understanding of the molecular mechanisms of FCR resistance/susceptibility in wheat and will benefit further efforts for the genetic improvement of disease resistance.

Salicylic acid positively regulates maize defenses against lepidopteran insects

In response to insect attack,plants use intricate signaling pathways,including phytohormones,such as jasmonate(JA),ethylene(ET),and salicylic acid(SA),to activate defenses.Maize(Zea mays)is one of the most important staple food crops around the world.Previous studies have shown that the JA and ET signaling play important roles in maize defense against insects,but little is known about whether and how SA regulates maize resistance to insect herbivores.In this study,we ectopically expressed the NahG(salicylate hydroxylase)gene in maize plants(NahG maize)to block the accumulation of SA.It was found that compared with the wild-type(WT)maize,the NahG-maize exhibited decreased resistance to the generalist insects Spodoptera litura and Spodoptera frugiperda and the specialist Mythimna separata,and the compromised resistance in the NahG maize was associated with decreased levels of defensive metabolites benzoxazinoids(Bxs)and chlorogenic acid(CA).Quantification of simulated S.litura feedinginduced JA,JA-isoleucine conjugate(JA-Ile),and ET in the WT and NahG maize indicated that SA does not regulate JA or JA-Ile,but positively controls ET.We provide evidence suggesting that the SA pathway does not crosstalk with the JA or the ET signaling in regulating the accumulation of Bxs and CA.Transcriptome analysis revealed that the bHLH,ERF,and WRKY transcription factors might be involved in SAregulated defenses.This study uncovers a novel and important phytohormone pathway in maize defense against lepidopterous larvae.

Horizontal transfer and evolution of the biosynthetic gene cluster for benzoxazinoids in plants

Benzoxazinoids are a class of protective and allelopathic plant secondary metabolites that have been identified in multiple grass species and are encoded by the Bx biosynthetic gene cluster(BGC)in maize.Data mining of 41 high-quality grass genomes identified complete Bx clusters(containing genes Bx1–Bx5 and Bx8)in three genera(Zea,Echinochloa,and Dichanthelium)of Panicoideae and partial clusters in Triticeae.The Bx cluster probably originated from gene duplication and chromosomal translocation of native homologs of Bx genes.An ancient Bx cluster that included additional Bx genes(e.g.,Bx6)is presumed to have been present in ancestral Panicoideae.The ancient Bx cluster was putatively gained by the Triticeae ancestor via horizontal transfer(HT)from the ancestral Panicoideae and later separated into multiple segments on different chromosomes.Bx6 appears to have been under less constrained selection compared with the Bx cluster during the evolution of Panicoideae,as evidenced by the fact that it was translocated away from the Bx cluster in Zea mays,moved to other chromosomes in Echinochloa,and even lost in Dichanthelium.Further investigations indicate that purifying selection and polyploidization have shaped the evolutionary trajectory of Bx clusters in the grass family.This study provides the first candidate case of HT of a BGC between plants and sheds new light on the evolution of BGCs.

9,10-KODA,an α-ketol produced by the tonoplastlocalized 9-lipoxygenase ZmLOX5,plays a signaling role in maize defense against insect herbivory

13-Lipoxygenases(LOXs)initiate the synthesis of jasmonic acid(JA),the best-understood oxylipin hormone in herbivory defense.However,the roles of 9-LOX-derived oxylipins in insect resistance remain unclear.Here,we report a novel anti-herbivory mechanism mediated by a tonoplast-localized 9-LOX,ZmLOX5,and its linolenic acid-derived product,9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid(9,10-KODA).Transposon-insertional disruption of ZmLOX5 resulted in the loss of resistance to insect herbivory.lox5 knockout mutants displayed greatly reduced wound-induced accumulation of multiple oxylipins and defense metabolites,including benzoxazinoids,abscisic acid(ABA),and JA-isoleucine(JA-Ile).However,exogenous JA-Ile failed to rescue insect defense in lox5 mutants,while applications of 1 mM 9,10-KODA or the JA precursor,12-oxo-phytodienoic acid(12-OPDA),restored wild-type resistance levels.Metabolite profiling revealed that exogenous 9,10-KODA primed the plants for increased production of ABA and 12-OPDA,but not JA-Ile.While none of the 9-oxylipins were able to rescue JA-Ile induction,the lox5 mutant accumulated lower wound-induced levels of Ca^(2+),suggesting this as a potential explanation for lower wound-induced JA.Seedlings pretreated with 9,10-KODA exhibited rapid or more robust woundinduced defense gene expression.In addition,an artificial diet supplemented with 9,10-KODA arrested fall armyworm larvae growth.Finally,analysis of single and double lox5 and lox10 mutants showed that ZmLOX5 also contributed to insect defense by modulating ZmLOX10-mediated green leaf volatile signaling.Collectively,our study uncovered a previously unknown anti-herbivore defense and hormonelike signaling activity for a major 9-oxylipin α-ketol.

ZmMYC2s play important roles in maize responses to simulated herbivory and jasmonate

Both herbivory and jasmonic acid(JA)activate the biosynthesis of defensive metabolites in maize,but the mechanism underlying this remains unclear.We generated maize mutants in which ZmMYC2a and ZmMYC2b,two transcription factor genes important in JA signaling,were individually or both knocked out.Genetic and biochemical analyses were used to elucidate the functions of ZmMYC2 proteins in the maize response to simulated herbivory and JA.Compared with the wild-type(WT)maize,the double mutant myc2ab was highly susceptible to insects,and the levels of benzoxazinoids and volatile terpenes,and the levels of their biosynthesis gene transcripts,were much lower in the mutants than in the WT maize after simulated insect feeding or JA treatment.Moreover,ZmMYC2a and ZmMYC2b played a redundant role in maize resistance to insects and JA signaling.Transcriptome and Cleavage Under Targets and TagmentationSequencing(CUT&Tag-Seq)analysis indicated that ZmMYC2s physically targeted 60%of the JAresponsive genes,even though only 33%of these genes were transcriptionally ZmMYC2-dependent.Importantly,CUT&Tag-Seq and dual luciferase assays revealed that ZmMYC2s transactivate the benzoxazinoid and volatile terpene biosynthesis genes IGPS1/3,BX10/11/12/14,and TPS10/2/3/4/5/8 by directly binding to their promoters.Furthermore,several transcription factors physically targeted by ZmMYC2s were identified,and these are likely to function in the regulation of benzoxazinoid biosynthesis.This work reveals the transcriptional regulatory landscapes of both JA signaling and ZmMYC2s in maize and provides comprehensive mechanistic insight into how JA signaling modulates defenses in maize responses to herbivory through ZmMYC2s.

The Auxin-Regulated Protein ZmAuxRPI Coordinates the Balance between Root Growth and Stalk Rot Disease Resistance in Maize

To optimize fitness, plants must efficiently allocate their resources between growth and defense. Although phytohormone crosstalk has emerged as a major player in balancing growth and defense, the genetic basis by which plants man age this balance remai ns elusive. We previously ide ntified a quantitative disease . resistance locus, qRfg2, in maize (Zea mays) that protects against the fungal disease Gibberella stalk rot. Here, through map-based cloning, we demonstrate that the causal gene at qRfg2 is ZmAuxRPI, which encodes a plastid stroma-localized auxin-regulated protein. ZmAuxRPI responded quickly to pathogen challenge with a rapid yet transient reduction in expression that led to arrested root growth but enhanced resista nee to Gibberella stalk rot and Fusarium ear rot. ZmAuxRPI was show n to promote the biosynthesis of indole-3-acetic acid (IAA), while suppressing the formation of benzoxazinoid defense compounds. ZmAuxRPI presumably acts as a resource regulator modulating indole-3-glycerol phosphate and/or indole flux at the branch point between the IAA and benzoxazinoid biosynthetic pathways. The concerted interplay between IAA and benzoxazinoids can regulate the growth-defense balance in a timely and efficient manner to optimize plant fitness.