In plants, a variety of stimuli trigger long-range calcium signals that travel rapidly along the vasculature to distal tissues via poorly understood mechanisms. Here, we use quantitative imaging and analysis to demonstrate that traveling calcium waves are mediated by diffusion and bulk flow of amino acid chemical messengers. We propose that wounding triggers release of amino acids that diffuse locally through the apoplast, activating the calcium-permeable channel GLUTAMATE RECEPTOR-LIKE 3.3 as they pass. Over long distances through the vasculature, the wound-triggered dynamics of a fluorescent tracer show that calcium waves are likely driven by bulk flow of a channel-activating chemical. We observed that multiple stimuli trigger calcium waves with similar dynamics, but calcium waves alone cannot initiate all systemic defense responses, suggesting that mobile chemical messengers are a core component of complex systemic signaling in plants.

The transcriptomics approach to study gene expression in root hairs from M. truncatula has shed light on the developmental events during rhizobial infection and the underlying hormone responses. This approach revealed the induction of several cyclins and an aurora kinase which suggests that the cell-division machinery plays a role in rhizobial infection. Changes in the cell cycle in plants are governed by hormones, in particular auxin and cytokinin. Through gene expression and genetic analyses, we have shown auxin plays a role during rhizobial infection. Here we provide further analysis of the data showing the induction of a set of cytokinin signaling components. These include genes encoding 2 cytokinin-activating enzymes, the cytokinin receptor CRE1, and 5 type-A cytokinin response regulators. We discuss the possible interactions between auxin and cytokinin signaling during the infection process. We also consider a potential role for cytokinin signaling in rhizobial attachment.

14The plasma membrane (PM) that lines plasmodesmata has a distinct protein and lipid 15 composition, underpinning specific regulation of these connections between cells. The 16 plasmodesmal PM can integrate extracellular signals differently from the cellular PM, but it 17 is not known how this specificity is established or how a single stimulus can trigger 18 independent signalling cascades in neighbouring membrane domains. Here we have used the 19 fungal elicitor chitin to investigate signal integration and responses at the plasmodesmal PM. 20 We found that the plasmodesmal PM employs a receptor complex composed of the LysM 21 receptors LYM2 and LYK4 which respectively change their location and interactions in 22 response to chitin. Downstream, signalling is transmitted via a specific phosphorylation 23 signature of an NADPH oxidase and localised callose synthesis that causes plasmodesmata 24 closure. This demonstrates the plasmodesmal PM deploys both plasmodesmata-specific 25 components and differential activation of PM-common components to independently 26 integrate an immune signal. 27 28 93that plasmodesmata are regulated independently of other immune responses, suggesting that 94 there is a critical requirement for a cell to finely tune connectivity to its neighbours. 95 4 Results 96Chitin-triggered plasmodesmata closure is dependent on LYK4 and LYK5 97 We previously identified that LYM2 is a GPI-anchored, LysM receptor protein that is 98 resident in the plasmodesmal PM (Faulkner et al., 2013). As LYM2 has no intracellular 99 domains we reasoned that it must interact with other proteins to initiate downstream signals 100 that result in plasmodesmal responses. Ligand perception by LysM RKs and RPs often 101 involves multiple members of the LysM protein family: chitin perception in rice involves 102 both the RP CHITIN ELICITOR BINDING PROTEIN (OsCEBiP) and the RK CHITIN 103 ELICITOR RECEPTOR KINASE 1 (OsCERK1) (Kaku et al., 2006; Hayafune et al., 2014); 104 peptidoglycan perception in Arabidopsis involves the RK CERK1, and RPs LYM1 and 105 LYM3 (Willmann et al., 2011); and PM chitin perception in Arabidopsis involves CERK1 106 (also called LYK1) and the RKs LYK4 and LYK5 (Cao et al., 2014). Thus, we hypothesised 107 that LYM2 might partner with a LysM RK for signalling. The Arabidopsis LysM RK family 108 consists of 5 members: CERK1/LYK1, LYK2, LYK3, LYK4 and LYK5. To narrow down 109 plasmodesmata signalling candidates we screened publicly available data sets for LYK gene 110 expression. Comparing data sets from seedlings (GSE74955, Yamada et al., 2016; 111 GSE78735, Hillmer et al., 2017) and mature leaves (eFP browser, Winter et al. 2007) we 112 identified variable expression patterns for the LYK family members (Fig. S1). Thus, we 113 performed RT-PCR to identify members of the family expressed in mature Arabidopsis 114 leaves where we assay for and detect LYM2 function. Only transcripts from CERK1, LYK3, 115LYK4 and LYK5 were detected in mature leaves grown in our conditions, eliminating LYK2 116 f...