Silicon modulates multi-layered defense against powdery mildew in Arabidopsis

Silicon (Si) has been widely employed in agriculture to enhance resistance against pathogens in many crop plants. However, the underlying molecular mechanisms of Si-mediated resistance remain elusive. In this study, the Arabidopsis-powdery mildew pathosystem was employed to investigate possible defense mechanisms engaged for Si-mediated resistance. Because Arabidopsis lacks efficient Si transporters and thus is a low Si-accumulator, two heterologous Si influx transporters (from barley and muskmelon) were individually expressed in wild-type Arabidopsis Col-0 and a panel of mutants defective in different immune signaling pathways. Results from infection tests showed that while very low leaf Si content slightly induced salicylic acid (SA)-dependent resistance, high Si promoted PAD4-dependent but EDS1- and SA-independent resistance against the adapted powdery mildew isolate Golovinomyces cichoracearum UCSC1. Intriguingly, our results also showed that high Si could largely reboot non-host resistance in an immune-compromised eds1/pad4/sid2 triple mutant background against a non-adapted powdery mildew isolate G. cichoracearum UMSG1. Taken together, our results suggest that assimilated Si modulates distinct, multi-layered defense mechanisms to enhance plant resistance against adapted and no-adapted powdery mildew pathogens, possibly via synergistic interaction with defense-induced callose.


INTRODUCTION
Silicon (Si) is the second most abundant element on the earth. Although it is not considered to be an essential element for plant growth, Si has long been recognized as a "beneficial" or "quasi-essential" substance to plants, mainly due to its important role in plant nutrition, particularly under stressful conditions. Over the past 25 years, a plethora of studies with >800 publications have collectively shown that Si can effectively protect plants from abiotic stresses, including drought, salinity and metals toxicity, as well as biotic stresses caused by insect herbivores or various pathogens ranging from viruses, bacteria, fungi, It has been demonstrated that Si needs to be absorbed by plants through passive channel-type, selective Si transporters to realize its prophylactic effect (Ma 2010).
Intriguingly, Si accumulation varies over 100-fold in different plant species, ranging from 0.1% to 10% in dry weight (Epstein 1994). Based on the levels of Si accumulation, plants are divided into three categories: high Si (or active) accumulator, intermediate Si (or passive) accumulator and low Si (rejective) accumulator (Takahashi et al. 1990). Understandably, most studies on Si transport and its physiological roles in plants have been conducted with high accumulators, and to a lesser extent, intermediate accumulators. The ability of a particular plant for accumulation of Si is determined by the transport efficiency of its Si influx transporters. The first identified Si influx transporter (OsLsi1) in higher plants was found in rice in 2006 (Ma et al. 2006). Since then, many Si transporters homologous to on published data generated with distinct pathosystems, two types of mechanisms have been proposed to explain how Si might act in plant cells. The first type conforms to the "apoplastic obstruction hypothesis" recently proposed by Coskun and colleagues based on many observations that Si is deposited on plant cell surface or in the apoplastic space whereby it strengthens physical barriers that can limit entry of pathogens and/or delivery of pathogen molecules (Coskun et al. 2019). For example, Si can accumulate and deposit beneath the cuticle of rice leaves to reduce penetration of rice blast Magnaporthe grisea (Yoshida 1965) and Si can also polymerize in specialized cells and cellular structures of some species (particularly grasses), such as leaf silica and long cells, and spikelet hairs and papillae to strengthen the physical barriers to prevent pathogen entry (Rafi et al. 1997). For the second type of mechanism, some studies have suggested that Si may be able to potentiate Thus, Si appears to promote both physical and chemical barriers against powdery mildew (and possibly other) pathogens. However, the genetic and molecular basis of Si's action largely remained unexplored until a recent genetic study in which transgenic Arabidopsis expressing a heterologous Si transporter was exploited to address this question.
Vivancos and colleagues (2015) expressed the wheat Si transporter (TaLsi1) in Arabidopsis wild-type Col-0 and mutants that are defective in SA-signaling (due to the loss of PAD4) or biosynthesis (due to the loss of SID2). They showed that TaLsi1-expressing Col-0 plants were able to accumulate high levels of Si and hence exhibited enhanced resistance to powdery mildew. Interestingly, they found that pad4 and sid2 mutant plants transgenic for TaLsi1 also showed similar levels of resistance when Si was supplied. These results suggested that Si, when above a threshold level, is able to activate disease resistance via an unknown mechanism(s) that is independent of SA signaling (Vivancos et al. 2015).
In this study, we employed the Arabidopsis-powdery mildew pathosystem to further investigate the molecular mechanisms of Si-mediated disease resistance in plants. Through the use of Arabidopsis genetic mutants, transgene expression of heterologous Si transporters and a more stringent control of Si supply, we found that Arabidopsis plants containing either very low or very high Si content displayed enhanced resistance to powdery mildew.
Interestingly, we found that whereas very low Si-induced resistance is SA-dependent, high Si-mediated resistance is indeed independent of SA signaling but surprisingly requires PAD4.
Moreover, we also found that high Si can effectively reboot penetration resistance in a SA signaling-defective and PAD4-ablated Arabidopsis mutant that is otherwise fully susceptible to a non-adapted powdery mildew isolate. Thus, our results present genetic evidence for the role of Si in multi-layered plant defense mechanisms, which should help clarify the current confusion and controversy over the molecular mechanisms underlying Si's positive role in plant disease resistance against fungal pathogens.

RESULTS
Plants with either very low or very high endogenous Si display enhanced resistance 6 against powdery mildew In our pilot experiment, we surprisingly found that Arabidopsis thaliana wild-type Col-0 plants became slightly more susceptible to an adapted powdery mildew isolate Golovinomyces cichoracearum (Gc) UCSC1 when irrigated water containing 1.7 mM Silicon. This is an equivalent concentration of Si that provided remarkable protection against B. graminis f. sp. tritici in wheat (Chain et al. 2009) compared to plants without Si ( Figure S1).
To reliably assess the role of Si in modulating plant defense mechanisms, we decided to use perlite irrigated with deionized pure water containing Si and a controlled amount of fertilizer to grow Arabidopsis plants (Xiao et al. 2003). We first tested eight-week-old wild-type Col-0 plants with Gc UCSC1. Intriguingly, we found that Col-0 plants irrigated with nutrient solution without Si (-Si) showed enhanced disease resistance, which was visible by the naked eye when compared with plants irrigated with water and fertilizer plus 1.0 mM Si (+Si) ( Figure 1A). Quantification of fungal spore production at 10 days post-inoculation (dpi) showed that plants of Col-0/-Si produced ~30% less spores than plants of Col-0/+Si ( Figure   1B). Leaf Si content in Col-0/-Si plants was 1.60 mg g -1 dry leaf tissue, whereas Col-0/+Si plants had slightly but significantly higher leaf Si content (2.38 mg g -1 ; ~48% increase) ( Figure 1C). These results reinforce the notion that Arabidopsis was a low Si-accumulator Col-0/+Si ( Figure 3B). By contrast, plants of the remaining genotypes that are defective in SA signaling or biosynthesis displayed no significant difference between -Si and +Si-treated plants ( Figure 3B). Quantification of fungal spore production supported the visual phenotypes ( Figure 3C). DAB staining showed that those mutants defective in SA-Signaling or biosynthesis (as represented by pad4) showed no H 2 O 2 accumulation in PM-invaded epidermal cells of either -Si or +Si-treated plants ( Figure 3A).
To further determine if very low leaf Si content somehow constitutively activates a SA-dependent defense mechanism, we also measured leaf SA and JA levels of the Col-0 plants before and after infection. We found that Col-0/-Si plants indeed had slightly but significantly higher total SA levels at 0 and 3 dpi compared to Col-0/+Si plants ( Figure 3D), whereas there was no significant difference in JA levels between Col-0/-Si and Col-0/+Si plants ( Figure 3E). Thus, together our data suggest that very low leaf Si content ectopically activates SA-dependent basal defense against powdery mildew.

High Si-conditioned resistance is SA-independent but PAD4-dependent
To investigate if high Si-conditioned stronger resistance has the same or a distinct mechanistic basis, we first wondered if the enhanced resistance in transgenic plants of Next, we introduced the same DNA constructs for expression of the two heterologous Si transporters into seven single Arabidopsis mutants (i.e. eds1, pad4, sid2, coi1, ein2), one 9 double (eds1pad4) and one triple (eds1pad4sid2) mutant. Measurement of Si content showed that expression of HvLsi1 or CmeLSi1 resulted in elevation of leaf Si content in the backgrounds of all of these mutants ( Figure S2), similar to that in Col-0 (Figure 2A), indicating that none of these immunity-related mutations interferes with Si uptake. We then grew plants of the above described representative transgenic lines under -Si or +Si conditions for seven weeks and then inoculated them with Gc UCSC1 and assessed their disease reaction phenotypes. As shown in Figure 5A&B, all transgenic lines without Si supplement exhibited disease susceptibility phenotypes as expected based on their genotypes (i.e. those SA-pathway defective mutants were more susceptible than Col-0 and those defective in JA or ET pathways). Strikingly, for the transgenic plants supplemented with Si, only plants of those genotypes that contain the pad4 mutation, i.e. pad4, eds1pad4 and eds1pad4sid2 did not show enhanced disease resistance relative to their counterparts without Si supplement ( Figure 5A-D). These results indicate that high Si content-mediated resistance is EDS1-, SA-and JA/ET-independent but PAD4-dependent.
To evaluate the molecular attributes of the PAD4-dependent mechanism activated by high Si content, we first used qRT-PCR to measure the expression levels of PR1 in plants of HvLsi1-Col-0 plants with or without Si treatment at 0, 3, 5 dpi with Gc UCSC1. We found that PR1 in HvLsi1-Col-0/+Si plants was highly expressed before powdery mildew infection (0 dpi) and had no or only slight increase at 3 or 5 dpi ( Figure S3A). This observation is in agreement with H 2 O 2 production independent of powdery mildew infection in HvLsi1-Col-0/+Si plants ( Figure 4D). Next, we examined if high leaf Si content can increase PAD4 expression. Interestingly, we found that although PAD4 was induced to higher levels by powdery mildew infection at 3 and 5 dpi, no significant difference was detected between -Si and +Si plants ( Figure S3B). This result suggests that high-level Si may not impact transcription of PAD4 but rather augment certain functionality of PAD4 via an unknown post-transcriptional mechanism, thereby activating this defense pathway. Not surprisingly, we detected no significant difference in PDF1.2 expression between -Si and +Si plants before and after powdery mildew infection ( Figure S3C), which was consistent with the observation that no significant phenotypic difference was found between transgenic lines of Col-0 and those of coi1 ( Figure 5). We also measured PMR4 expression under different Si conditions given that the callose formation and deposition may coordinate with biological silicification in Arabidopsis (Brugié re and Exley 2017). The result showed that Si content had little impact on PMR4 expression ( Figure S3D). To further test if the PAD4-dependent, high Si-conditioned resistance is influenced by SA or JA biosynthesis, we also measured the total SA and JA levels in the transgenic -Si and +Si plants and found that levels of total SA were significantly higher in HvLsi1-Col-0/+Si plants compared to the plants of the same line with Si-treatment before and after powdery mildew infection ( Figure S4A). As expected, the levels of total SA were significantly lower in transgenic eds1, pad4, and particularly sid2 lines in comparison with those of transgenic Col-0 under either -Si or +Si conditions ( Figure S4 A,C,E,G).
Interestingly, compared with the respective -Si plants, JA levels were significantly higher in This resistance mechanism is obviously different from that of the PAD4-dependent resistance mediated by high Si against the well-adapted Gc UCSC1 isolate where no gross difference in hyphal growth of sporelings at 2 dpi was observed between -Si and +Si plants ( Figure S6).

DISCUSSION
Si has long been known to increase disease resistance in plants. The suppressive effect of Si on powdery mildew was first reported in 1983 (Miyake and Takahashi 1983). However, the molecular mechanisms underlying Si-mediated resistance remain largely elusive and even controversial still today. In this study, by using the Arabidopsis-powdery mildew pathosystem, we collected genetic evidence to demonstrate that Si affects different layers of plant defense in a dosage-dependent manner, providing novel mechanistic insights into Si's prophylactic role in plants against fungal pathogens.

12
A few previous studies on how Si might affect basal resistance to adapted powdery mildew using Arabidopsis Col-0 wild-types did not generate consistent results (Ghanmi et al.

2004; Fauteux et al. 2006; Vivancos et al. 2015)
. One contributing factor might be that different types or even batches of commercial soil for growing Arabidopsis may vary in Si content. This prompted us to use perlite as soil medium (Xiao et al. 2003) in this study to enable tighter control of Si supplement, which led to our observation that Col-0 plants with a very low leaf Si content (<2.0 mg g -1 ) displayed slightly but visually discernable and statistically significant enhanced resistance against the adapted powdery mildew Gc UCSC1 ( Figure 1). The resistance was found to be associated with H 2 O 2 accumulation in the invaded cells and require intact SA-signaling ( Figure 3). Interestingly, a previous study also showed that oat plants deprived for Si exhibited higher phenylalanine ammonia lyase activity compared to those with normal Si supply, which led to a speculation that lack of Si may activate a compensatory mechanism resulting in better resistance to powdery mildew penetration resistance (Carver et al. 1998). Hence, we speculate that Si content below a certain threshold showed that the high Si-enhanced resistance was not affected by the loss of PAD4 or SID2, which differs from our result that high Si-mediated resistance is PAD4-dependent ( Figure 3).
Our conclusion was inferred from the infection phenotypes of wild-type, single, double and triple pad4-containing transgenic lines expressing one of the two selected heterologous Si transporters using very even conidia inoculation (see Methods for details). It is possible that the discrepancy concerning PAD4 might have been caused by the differences in powdery mildew inoculation and/or growth conditions. Nevertheless, our results corroborated with their conclusion that high Si enhances SA-independent resistance to powdery mildew. The PAD4-dependence of high Si-mediated resistance is particularly interesting, because it suggests that high Si may activate or potentiate an intracellular defense mechanism beyond apoplastic obstruction (Coskun et al. 2019). Moreover, it also suggests that this PAD4-dependent mechanism is distinct from that activated by PAD4 and its partner EDS1 in In summary, results from this study implicated Si in three unexpected and distinct defense mechanisms that lead to enhanced resistance to PM: (1) very low Si-triggered SA-dependent defense; (2) high Si-mediated PAD4-dependent defense; and (3) high Si-boosted penetration resistance. How to make sense of these seemingly distinct mechanisms?
Information from previous reports (see below) and this study as a whole appears to support the "Si-callose synergy theory" (Brugié re and Exley 2017) which may offer a plausible explanation for our observations. First, Si is deposited to callose-rich papillae thereby playing a positive role in restricting cell-wall penetrating fungal pathogens such as powdery mildew Lastly, PAD4-dependent resistance to aphids is also associated with increased callose deposition (Rashid et al. 2017), similar to Si-mediated resistance to insects (Yang et al. 2018) or even nematodes (Zhan et al. 2018), despite that in the latter cases whether resistance is PAD4-dependent remains to be tested. Therefore, it is possible that PAD4 (but not EDS1) may play a critical role in the deposition of a basal level of PMR4-dependent callose to papillae, thus explaining the PAD4-dependence of high Si-mediated resistance against powdery mildew pathogens. Combining all the above information, we developed a schematic diagram to summarize our genetic data on Si and hypothesize that deposition of Si, along with callose, to the papilla in plant cells enhances defense against powdery mildew infection in three different scenarios ( Figure S9). Future research is needed to investigate whether and how Si and callose may synergize with each other to fortify cell wall-based defense against fungal invasion, how exactly PAD4 regulates this defense mechanism, whether Si-mediated resistance to insects also requires a PAD4-regulatory node, and whether this mechanistic model is also applicable to medium-and high-Si-accumulating plants.

Plant lines and growth conditions
All mutants used in this study were in the Arabidopsis thaliana accession Col-0 background.

DNA constructs and generation of transgenic lines
To make Arabidopsis absorb more Si from soil medium, we generated stable transgenic lines  Table S1). The DNA fragments were cloned into pENTR/D-TOPO (Thermo Fisher Scientific Inc.) and shuttled to the Gateway Compatible binary vector pEarleyGate100. After DNA sequence confirmation, the constructs were introduced into Arabidopsis plants via Agrobacterium-mediated transformation using the A. tumefaciens strain GV3101. At least 20 independent T1 transgenics were obtained for each DNA construct/genotype combination. T2 progenies (24 plants) of at least ten T1 lines were grown under -Si and +Si conditions and inoculated with powdery mildew to visually assess the infection phenotypes. T3 generations derived from three T1 independent lines were used to confirm the infection phenotypes, and one representative homozygous T3 line for each genotype was used for comparative and quantitative analysis with other relevant genotypes.

Pathogen infection and quantification of disease phenotypes
Adapted powdery mildew isolate Golovinomyces cichoracearum (Gc) UCSC1 was maintained on pad4 plants and the non-adapted isolate Gc UMSG1 was maintained on sow thistle plants (Wen et al. 2010). Inoculation and visual scoring of disease reaction phenotypes and spore quantification were done as previously described (Zhang et al. 2018). Briefly, for quantification of disease susceptibility, five or six duplicate leaf samples (each consisting of ~120 mg leaves) collected from 12 plants of each representative T3 line at 10 dpi were used to quantify the level of sporulation. A spore suspension (or 10x dilution if the genotype was very susceptible) of each sample, which was made by vortexing the leaves in a 50 ml falcon tube containing 10 ml of H 2 O + 0.02% Silwet L-77 (Lehle seeds, USA) for one minute, was used for spore counting using Luna TM Automated Cell Counter (Logos biosysems). Spore counts were normalized to the fresh weight of the corresponding leaf samples. All infection trials with T3 generations were repeated three times with similar results, and data from one experiment were presented.

Detection of H 2 O 2 , callose deposition and fungal structures
The detection of H 2 O 2 accumulation in leaf tissues by 3,3-diaminobenzidine (DAB) staining was modified from Thordal-Christensen et al. (1997). Inoculated leaves were excised at the base of the petiole, placed in 1 mg ml -1 DAB (Sigma), and incubated for 6 h at 25°C with illumination. Fungal structures in inoculated leaves were visualized with 0.25% Trypan blue staining solution (Xiao et al. 2003). Callose deposition at the fungal penetration sites (i.e. papillae) and around the haustorium was detected by aniline blue staining. Leaves were cleared in a solution containing ethanol, water, acetic acid, and glycerol (8:1:1:1) for 48 hours at 37 ℃ with one change of the solution. The cleared leaves are then stained with 0.01% aniline blue in an aqueous solution containing 150 mM KH 2 PO 4 (PH 9.5) for 4 hours.
Callose deposition was visualized by fluorescence microscopy.

Measurement of Si content in Arabidopsis leaves
Leaf Si content was determined ten days after treatment with +Si or -Si nutrient solution using seven or eight-week-old plants. All rosette leaves from five +Si or -Si plants per sample were oven dried at 65˚C for 72 h, and ground into a fine powder using a mortar and 18 pestle before measurement of Si concentration by colorimetric analysis using 0.1 g alkali-digested leaf tissue powder (Frantz et al. 2008). Three duplicated samples were processed for each genotype-treatment combination and Si content was calculated, adjusted for dry weight of leaf tissues used, and presented as mg Si dioxide per gram dry matter.  Supplemental Table S1.

Measurement of levels of SA and JA
Five leaf samples (~150 mg each) per genotype-treatment were harvested at 0, 3 and 5 dpi with Gc UCSC1 for determining levels of both SA and JA as previously described (Floková et al. 2014), with some modifications. For each experiment, detection was performed with three biological replicates per treatment. The leaf tissues in a 1.5 ml tube were added with 1 ml of 50% ethanol containing the internal standards (vanillic acid and dihydro jasmonic acid), four steel balls (diameter 5 mm), and then shaken for 5×1 minute in a TissueLyser II (QIAGEN) at 25Hz with one min pause between every min before centrifuged at 20,000g for 10 mins. The supernatant was analyzed with a Waters Acquity UPLC system equipped with a Waters LCT Premiere XE ESI-TOF mass spectrometer. The detailed method is described in Methods S1.